JP2018521130A - Methods and compositions for treating drug diseases and conditions - Google Patents

Abstract

Methods and compounds useful for treating drug-induced diseases and conditions are provided. As a treatment for a drug-borne disease or condition, a method is provided for administering one or more MCJ inhibitor compounds to reduce MCJ polypeptide activity in a cell, tissue, and / or subject. [Selection] Figure 1B

Description

RELATED APPLICATIONS This application is filed under US Provisional Application No. 62 / 190,803 filed July 10, 2015 and US filed April 19, 2016 under 35 USC 119 (e). No. 62 / 324,584, all of which are hereby incorporated by reference in their entirety.

Government Rights This invention was made with government support under R21 AI094027 awarded by the National Institutes of Health. The government has certain rights in the invention.

The present invention relates, in part, to methods and compounds useful for treating toxic and drug diseases and conditions.

  Cirrhosis is a global health problem affecting up to 1% of the population and a major risk factor for progression to hepatocellular carcinoma (HCC), one of the most frequent fatal solid organ tumors. is there. Several pathogenic factors contribute to the development of cirrhosis, but hepatitis C, fatty liver and alcohol dependence are the most common causes. Cirrhosis is characterized by an advanced stage of liver fibrosis. Currently, liver transplantation is one of the foundations in the management of cirrhosis, but its application is limited by strict selection criteria, high costs and a lack of donor transplant tissue.

  Liver injury can also be induced by drug accumulation. Drug-induced liver injury (DILI) includes medicinal herbs, plants, and dietary supplements as well as injuries caused by numerous drugs. Drug-induced injury mechanisms can include covalent binding of drugs to cellular proteins resulting in immune damage, inhibition of cellular pathways, blockage of cell transport pumps, induction of apoptosis, mitochondrial dysfunction, and the like. Overdose of acetaminophen (Tylenol) leading to hepatotoxic effects has been reported to be a major cause of acute liver failure in the United States and the United Kingdom. The FDA has proposed limiting hepatic toxicity of acetaminophen by reducing its therapeutic index and minimizing combination therapy.

  According to one aspect of the invention, methods are provided for treating toxic and drug diseases and conditions in the liver and other organs and organ systems in a subject. The method includes administering to the cell and / or subject in need of such treatment an MCJ inhibitor compound in an amount effective to treat the toxic and drugous disease or condition in the cell and / or subject, respectively. .

  According to one aspect of the invention, methods for treating drug-induced diseases and conditions in a subject are provided. The method includes administering the MCJ inhibitor compound to a subject in need of such treatment in an amount effective to treat the drug-induced disease or condition in the subject. In some embodiments, the MCJ modulating compound is an MCJ inhibitory compound that reduces MCJ polypeptide activity in the subject. In some embodiments, reducing MCJ polypeptide activity comprises reducing one or more of MCJ polypeptide levels or activities. In certain embodiments, the drug-borne disease or condition is one or more of drug-induced liver disease or condition and kidney disease or condition, heart disease or condition, and cardiovascular disease or condition. In some embodiments, the disease or condition is an acute disease or condition. In some embodiments, the disease or condition is a chronic disease or condition. In certain embodiments, the MCJ modulating compound comprises one or more of an MCJ molecule, an anti-MCJ polypeptide antibody or functional fragment thereof, and a small molecule MCJ inhibitor. In some embodiments, the MCJ modulating compound further comprises a targeting agent, optionally a mitochondrial targeting agent. In some embodiments, the MCJ molecule is a mutant MCJ polypeptide or a polynucleotide encoding a mutant MCJ polypeptide. In some embodiments, the small molecule MCJ inhibitor is a small interfering RNA molecule (siRNA), a small hairpin RNA (shRNA) molecule, an antisense DNA oligo, a small guide RNA (sgRNA) molecule, a transcriptional activator-like effector. Nuclease (talen) molecule. In certain embodiments, the siRNA molecule comprises the nucleic acid sequence described herein as SEQ ID NO: 7. In certain embodiments, the siRNA molecule comprises the nucleic acid sequence set forth herein as SEQ ID NO: 21. In some embodiments, the MCJ modulating compound is administered in a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, optionally a carrier agent, a delivery agent, a labeling agent, and a targeting agent. One or more of. In some embodiments, the carrier agent comprises one or more of nanocarriers, cell penetrating peptides, polymers, dendrimers, siRNA bioconjugates, and lipid-based siRNA carriers. In certain embodiments, the pharmaceutical composition further comprises a drug known or suspected to induce a drug-borne disease or condition. In some embodiments, the drug-borne disease or condition is drug-induced cirrhosis, liver fibrosis, venous obstructive liver disease, idiosyncratic toxicity, Budd-Chiari syndrome, liver injury, kidney injury, drug allergy, acute kidney Includes one or more of injury (AKI), fulminant hepatitis, cholestasis, cardiotoxicity, and alcohol consumption. In some embodiments, the drug induced disease or condition drug comprises one or more of ethanol, a pharmaceutical agent, and a biological agent. In some embodiments, the inducing drug enters the subject by means of ingestion, optionally including one or more of inhalation, injection, absorption, infusion, infusion, drinking, and feeding. In certain embodiments, pharmaceuticals include statins, antidepressants, antibiotics, benzodiazepines, nicotinic acid, tacrine, aspirin, quinidine, NSAIDs (including but not limited to aspirin, indomethacin, ibuprofen, naproxen, piroxicam, nabumetone) Acetaminophen, phenytoin, isoniazid, diclofenac, augmentin (amoxicillin / clavulanic acid combination), minocycline, nitrofurantoin, fenofibrate, methamphetamine, amphetamine, erythromycin, chlorpromazine, cotrimoxazole (sulfamethoxax Sol and trimethoprim combination), amitriptyline, temazepam, diazepam, carbamazepine, ampicillin, rifampin, est Diols, captopril, oral contraceptive pills (oral contraceptives), anabolic steroids, disulfiram, vitamin A, haloperidol, imipramine, tetracycline, phenytoin, methotrexate, amiodarone, methyldopa, chemotherapeutic agents, contrast agents, thiazines (phenothiazines, Including, but not limited to, chloramphenicol, digoxin, digitoxin, oxazepam, phenobarbital, quinidine, vancomycin, theophylline, verapamil, interferon (including but not limited to interferon beta 1a), and warfarin. In certain embodiments, the biological agent is an herbal extract. In certain embodiments, the herbal extract comprises one or more of ephedula (Ma Huang), Kava Kava, chaparral, valerian, cypress extract, and Kava leaves. In some embodiments, the drug-borne disease or condition is overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia, steatosis (e.g., (Hepatic steatosis, cardiac steatosis, renal steatosis, myolipidosis), β-lipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; liver disease, hepatitis, hepatitis, steatohepatitis, type C Hepatitis, genotype 3 hepatitis C, alpha 1-antitrypsin deficiency, acute fatty liver of pregnancy, Wilson disease; kidney disease; heart disease, hypertension, ischemia, heart failure, cardiomyopathy; addiction; HIV; neurodegenerative disease, Parkinson Not a disease, Alzheimer's disease; or a metabolic disease or condition of cancer. In some embodiments, the subject is overweight, gained weight, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia, steatosis (eg, hepatic steatosis, Cardiac steatosis, renal steatosis, muscle steatosis), β-lipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; liver disease, hepatitis, hepatitis, steatohepatitis, hepatitis C, genotype 3 hepatitis C, alpha 1-antitrypsin deficiency, acute fatty liver of pregnancy, Wilson disease; kidney disease; heart disease, hypertension, ischemia, heart failure, cardiomyopathy; addiction; HIV; neurodegenerative disease, Parkinson's disease, Alzheimer's disease And not having one or more of the metabolic diseases or conditions of cancer. In some embodiments, MCJ-modulating compounds are administered to a subject one or more before, during and after ingestion by a subject of a drug suitable for inducing a drug-induced disease or condition.

  According to another aspect of the invention, a method for reducing a drug-borne disease or condition in a cell is provided. The method includes contacting a cell with an MCJ inhibitor compound in an amount effective to reduce MCJ polypeptide activity in the cell. In certain embodiments, reducing MCJ polypeptide activity comprises reducing one or more of the level or function of MCJ polypeptide in the cell. In some embodiments, the cell is in vitro, ex vivo, or in vitro. In some embodiments, the in vivo cells are present in the subject and contacting comprises administering to the subject an MCJ inhibitor compound. In certain embodiments, the MCJ inhibitor compound comprises an MCJ molecule, an anti-MCJ polypeptide antibody or functional fragment thereof, or a small molecule MCJ inhibitor. In some embodiments, the MCJ inhibitor compound further comprises a targeting agent, optionally a mitochondrial targeting agent. In some embodiments, the MCJ molecule is a mutant MCJ polypeptide or a polynucleotide encoding a mutant MCJ polypeptide. In certain embodiments, the small molecule MCJ inhibitor is an siRNA molecule. In some embodiments, the siRNA molecule comprises the nucleic acid sequence described herein as SEQ ID NO: 7. In some embodiments, the small molecule MCJ inhibitor molecule has a nucleic acid sequence described herein as SEQ ID NO: 21. In some embodiments, the cell is contacted with an MCJ inhibitor compound present in the pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, optionally, a carrier agent, a delivery agent, a label. Agent, and one or more of targeting agents. In some embodiments, the drug-induced disease or condition is one or more of drug-induced liver disease or condition and kidney disease or condition, drug-induced heart disease or condition. In certain embodiments, the drug-borne disease or condition is one or more of an acute disease or condition and a chronic disease or condition. In some embodiments, the carrier agent comprises one or more of nanocarriers, cell penetrating peptides, polymers, dendrimers, siRNA bioconjugates, and lipid-based siRNA carriers. In some embodiments, the pharmaceutical composition further comprises a drug known or suspected to induce a drug-borne disease or condition. In certain embodiments, the drug-related disease or condition is drug-induced cirrhosis, liver fibrosis, venous obstructive liver disease, idiosyncratic toxicity, Budd-Chiari syndrome, liver injury, kidney damage, drug allergy, acute kidney Includes one or more of disorders (AKI), fulminant hepatitis, cholestasis, cardiotoxicity, and alcohol consumption. In some embodiments, the drug induced disease or condition drug comprises one or more of ethanol, a pharmaceutical agent, and a biological agent. In some embodiments, a drug-induced disease or condition is induced in a cell by contacting the inducing drug with the cell. In certain embodiments, the medicament comprises statins, antidepressants, antibiotics, benzodiazepines, nicotinic acid, tacrine, aspirin, quinidine, NSAIDs (including but not limited to aspirin, indomethacin, ibuprofen, naproxen, pyroxamine, nabumetone). Acetaminophen, phenytoin, isoniazid, diclofenac, augmentin (amoxicillin / clavulanic acid combination), minocycline, nitrofurantoin, fenofibrate, methamphetamine, amphetamine, erythromycin, chlorpromazine, cotrimoxazole (sulfamethoxax Sol and trimethoprim combination), amitriptyline, temazepam, diazepam, carbamazepine, ampicillin, rifampin, est Diols, captopril, oral contraceptive pills (oral contraceptives), anabolic steroids, disulfiram, vitamin A, haloperidol, imipramine, tetracycline, phenytoin, methotrexate, amiodarone, methyldopa, chemotherapeutic agents, contrast agents, thiazines (phenothiazines, Including, but not limited to, chloramphenicol, digoxin, digitoxin, oxazepam, phenobarbital, quinidine, vancomycin, theophylline, verapamil, interferon (including but not limited to interferon beta 1a), and warfarin. In some embodiments, the biological agent is an herbal extract. In certain embodiments, the herbal extract comprises one or more of ephedula, birch, chaparral, valerian, cypress extract, and birch leaves. In certain embodiments, the drug-borne disease or condition is overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia, steatosis (e.g., (Hepatic steatosis, cardiac steatosis, renal steatosis, myolipidosis), β-lipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; liver disease, hepatitis, hepatitis, steatohepatitis, type C Hepatitis, genotype 3 hepatitis C, alpha 1-antitrypsin deficiency, acute fatty liver of pregnancy, Wilson disease; kidney disease; heart disease, hypertension, ischemia, heart failure, cardiomyopathy; addiction; HIV; neurodegenerative disease, Parkinson Not a disease, Alzheimer's disease; or a metabolic disease or condition of cancer. In some embodiments, the cells are overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia, steatosis (eg, hepatic steatosis, Cardiac steatosis, renal steatosis, muscle steatosis), β-lipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; liver disease, hepatitis, hepatitis, steatohepatitis, hepatitis C, genotype 3 hepatitis C, alpha 1-antitrypsin deficiency, acute fatty liver of pregnancy, Wilson disease; kidney disease; heart disease, hypertension, ischemia, heart failure, cardiomyopathy; addiction; HIV; neurodegenerative disease, Parkinson's disease, Alzheimer's disease And not having one or more of the metabolic diseases or conditions of cancer. In some embodiments, the cell is contacted with the MCJ modulating compound one or more before, during and after contact of the cell with a drug suitable for inducing a drug-induced disease or condition.

  According to yet another aspect of the invention, a composition comprising an MCJ inhibitor compound and a pharmaceutically acceptable carrier is provided. In certain embodiments, the composition further comprises one or more of a carrier agent, a delivery agent, a labeling agent, and a targeting agent. In some embodiments, the carrier agent comprises one or more of nanocarriers, cell penetrating peptides, polymers, dendrimers, siRNA bioconjugates, and lipid-based siRNA carriers. In some embodiments, the pharmaceutical composition further comprises a drug known or suspected to induce a drug-borne disease or condition. In certain embodiments, the MCJ inhibitor compound comprises a targeting agent, optionally a mitochondrial targeting agent. In some embodiments, the MCJ inhibitor compound comprises a small molecule MCJ inhibitor. In certain embodiments, the small molecule MCJ inhibitor is a small interfering RNA molecule (siRNA), a small hairpin RNA (shRNA) molecule, an antisense DNA oligo, a small guide RNA (sgRNA) molecule, a transcriptional activator-like effector. Nuclease (talen) molecule. In some embodiments, the small molecule MCJ inhibitor molecule has a nucleic acid sequence described herein as SEQ ID NO: 7. In some embodiments, the small molecule MCJ inhibitor molecule has a nucleic acid sequence described herein as SEQ ID NO: 21. In some embodiments, the composition comprises a drug suitable for inducing a drug-borne disease or condition in at least one of the cell and the subject, respectively, when the drug is contacted with the cell or delivered to the subject. Further included.

  The present invention is not intended to be limited to compositions or methods that must meet one or more of any described object or feature of the present invention. It is also important that the present invention is not limited to the exemplary or main embodiments described herein. Modifications and substitutions by one skilled in the art are considered to be within the scope of the present invention.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 is the amino acid sequence of the human DNAJ domain-containing protein MCJ described as GENBANK® accession number AAD38506.1:
MAARGVIAPVGESLRYAEYLQPSAKRPDADVDQQGLVRSLIAVGLGVAALAFAGRYAFRIWKPLEQVITETAKKISTPSFSSYYKGGFEQKMSRREAGLILGVSPSAGKAKIRTAHRRVMILNHPDKGGSPYVAAKINEAKDLLETTTKH
SEQ ID NO: 2 is the mRNA sequence (complete CDS) of human DNAJ domain-containing protein MCJ described as GENBANK® Accession No. AF126743.1:

SEQ ID NO: 3 is the amino acid sequence of a mouse DNAJ domain-containing protein:
MATGGGVTSRESLRYAEYLPPSAQRSDADIDHTAGRRLIAVGLGVAAVAFAGRYAFQIWKPLEQVITATARKISSPSFSSYYKGGFEQKMSKREASLILGVSPSAGKAKIRTAHKRIMILNHPDKGGSPYVASKINEAKDLLEASSKAN
SEQ ID NO: 4 is the amino acid sequence of a subfamily C human DnaJ (Hsp40) homolog described as GENBANK® accession number AAH95400.1:
MAARGVIAPVGESLRYAEYLQPSAKRPDADVDQQRLVRSLIAVGLGVAALAFAGRYAFRIWKPLEQVITETAKKISTPSFSSYYKGGFEQKMSRREAGLILGVSPSAGKAKIRTAHRRVMILNHPDKGGSPYVAAKINEAKDLLETTTKH
SEQ ID NO: 5 is the nucleotide sequence of a subfamily C human DnaJ (Hsp40) homolog described as GENBANK® accession number BC095400.1:

SEQ ID NO: 6 is the human MCJ cDNA sequence.

The following siRNA molecules are also called siMCJ oligos and are double-stranded RNA oligos.

SEQ ID NO: 7 human siRNA sequence gaagatttcaactcctagc.
SEQ ID NO: 8 human siRNA sequence ggcgagaagctggtcttattt.
SEQ ID NO: 9 human siRNA sequence gctaagattagaacagctcat.

SEQ ID NO: 10 human siRNA sequence gctcataggagagtcatgatt.
SEQ ID NO: 11 human siRNA sequence tttgggactctgcctatataa.
SEQ ID NO: 12 human siRNA sequence gttgcagctcttgcatttgca.

SEQ ID NO: 13 human siRNA sequence ctacgcatttcggatctggaa.
SEQ ID NO: 14 human siRNA sequence gcagggactggtaagaagttt.
SEQ ID NO: 15 human siRNA sequence gttgcagctcttgcatttgca.

SEQ ID NO: 16 human siRNA sequence cagataaaggtggatctcctt.
SEQ ID NO: 17 human siRNA sequence gctcataggagagtcatgatt.
SEQ ID NO: 18 human siRNA sequence gctaagattagaacagctcat.

SEQ ID NO: 19 human siRNA sequence gtttgatagctgtaggact.
SEQ ID NO: 20 human siRNA sequence tcacccagataaaggtgga.
SEQ ID NO: 21 human siRNA sequence gaagatttcaactcctagctt.

SEQ ID NO: 22 Mouse siRNA sequence gcgagaggctagtcttatt.
SEQ ID NO: 23 is the mouse MCJ cDNA sequence:
tcggagtcctgcagtgccatggctaccggtggcggcgtgacctccagagaggggctgcgctacgccgaatacctgcctccttctgcccaaaggtcggacgccgacatcgaccacacagcggggagaaggttgctagctgtaggactaggtgttgcagctgttgcatttgcaggtcgctatgcatttcagatctggaaacctctagaacaagtaatcacggcaacagcaaggaagatttcctctccaagcttttcatcctactataaaggaggattcgagcagaaaatgagtaagcgagaggctagtcttattttaggtgtaagcccatctgctggcaaggccaagattagaacagcacacaagagaattatgattttaaaccatccagacaaaggtggatctccttacttagcatccaaaataaatgaagcaaaagatttgctcgaagcatccagcaaagctaactgatgctaaaggactgtacataccgagggaaaatggaacaaacgcacagctgtaaaagtccttcagaagaatgtggcacgtggtcgtgttccatactgacccagtctgttttctgtcattaagtgtgcagcaataaaagcctggcagccttgcagccttggtctggcagggacttcatccgtcaaaaaaaaaaaaaaaaaaaaaaa

1A-B are microscopic images and graphs showing increased MCJ levels in livers from cirrhotic patients. The results of immunohistochemistry in the left panel of FIG. 1A show MCJ expression in the liver from a healthy control subject (NL) (n = 5), and the results of immunohistochemistry in the right panel of FIG. 1A show cirrhosis (hepatitis C) ) Shows MCJ expression in liver from patients with n = 16. FIG. 1B is a graph showing the level of MCJ expression determined for healthy and cirrhotic patients. The results show a statistically significant increase in the level of MCJ in cirrhotic liver. Figures 2A-B provide microscopic images and graphs showing the use of acetaminophen administration as a mouse model for drug-induced liver injury (DILI). Results are shown for wild type (WT) and MCJ knockout (KO) mice that received acetaminophen intraperitoneally. FIG. 2A shows the results after 48 hours and shows the presence of macrophages in liver sections examined by immunohistochemistry for the F4 / 80 macrophage marker. FIG. 2A (left panel) shows the results for WT and FIG. 2A (right panel) shows the results for MCJ KO. The right graph of FIG. 2B shows the levels of ALT and AST transaminases in the serum. Results showed that KO mice are more resistant to damage induced by acetaminophen in the liver. FIG. 2B left panel shows fewer F4 / 80 macrophage markers in KO mice. MCJ KO mice developed less inflammation than WT mice and had lower levels of transaminase. FIG. 2B (left panel) is representative of one mouse for each genotype, and FIG. 2B (right panel) shows values for n = 4 mice for each genotype. 3A-C are diagrams showing results from WT and MCJ KO mice that underwent biliary ligation surgery using a biliary ligation (BDL) mouse model for cirrhosis. FIG. 3A shows a microscopic image of the liver after 14 days. Tissue damage is indicated by the presence of macrophages (F4 / 80) by IHC as a marker of inflammation. The left panel of FIG. 3A is an image derived from one WT mouse, and the right panel of FIG. 3A is an image derived from one MCJ KO mouse. FIG. 3B is a graph showing F4 / 80 positive staining values for n = 4 mice for each genotype. FIG. 3C provides survival curves for WT and MCJ KO mice during bile duct ligation surgery. MCJ KO mice are more resistant to developing cirrhotic liver damage. 4A-B provide graphs showing that MCJ shRNA (shMCJ) protected primary hepatocytes from DCA-induced death. FIG. 4A shows oxygen consumption rate (OCR) values in primary hepatocytes from WT mice transfected with control (WT) or shMCJ (shMCJ) expression plasmids. Results showed that shMCJ treatment reduced OCR compared to WT. FIG. 4B shows caspase-3 (a marker for cell death) after treatment with 100 μM DCA in primary hepatocytes from WT mice transfected with control (WT) or shMCJ (shMCJ) expression plasmids. The results showed that treatment with shMCJ reduced caspase-3 activity compared to WT. Figures 5A-B provide microscopic images and graphs showing MCJ expression examined by immunohistochemistry followed by further quantification. FIG. 5A shows MCJ expression in livers from healthy control subjects (control) (n = 7) and patients with drug-induced liver injury (DILI) (n = 23) as determined by immunohistochemistry. FIG. 5B shows in control mice (n = 3) and in livers of mice treated with acetaminophen (APAP) for 24 or 48 hours as determined by Western blot analysis and the ratio of MCJ to GAPDH as a control. The expression of MCJ is shown. ^* Means statistically significant (p <0.05). ^** means statistically significant (p <0.01). 6A-C provide graphs from studies showing that MCJ protected hepatocytes from acetaminophen-induced mitochondrial dysfunction and death. 6A is a graph of ATP levels, FIG. 6B is a graph of mitochondrial ROS levels, and FIG. 6C is WT and MCJ KO primary hepatocytes treated with acetaminophen (10 mM) for 9 hours (n = 3). 2 is a graph showing cell death as measured by the TUNEL assay in FIG. ^* Means statistically significant (p <0.05), ^*** means statistically significant (p <0.001). FIG. 7 provides a blot of results showing that MCJ siRNA (siMCJ) can be used to disrupt MCJ expression in the liver in vivo. Wild-type mice were administered siMCJ (1.7 mg / Kg) in combination with Invivofectamine by intravenous injection. Each mouse was collected at the indicated time after dosing. Livers were collected and MCJ expression was determined by Western blot analysis. GADPH was used as a loading control. 8A-C provide blots, microscopic images, and graphs showing that administration of siMCJ prevented acetaminophen-induced liver injury. FIG. 8A shows in vivofectamine 3.0. Shows blots of results from wild-type mice that were given intravenous injections of siMCJ (1.7 mg / Kg) in combination with (siMCJ mice) or without siMCJ (control mice). Twenty hours later, mice received an intraperitoneal dose of acetaminophen (360 mg / Kg). Mice were collected 24 hours later and MCJ expression in the liver was determined by Western blot analysis. Each lane of the blot represents a liver from an individual mouse (n = 3). 8B-C show the results from mice (n = 5) treated and collected as described in FIG. 8A. FIG. 8B shows representative H & E staining from liver sections, and FIG. 8C shows the mean value of liver damage (n = 5). ^* , P <0.05. Statistical significance was determined by Student's t test. 9A-B are Western blots and graphs showing that treatment with siMCJ after acetaminophen prevented acetaminophen-induced liver injury. For the study, wild type mice (n = 3) were administered an intraperitoneal dose of acetaminophen (360 mg / Kg). After 24 hours, the mice were in vivofectamine 3.0. Intravenous injection of siMCJ (1.7 mg / Kg) in combination with (siMCJ mice) or without siMCJ (control mice). Mice were collected 24 hours later (total 48 hours after acetaminophen overdose) and liver and serum collected. FIG. 9A shows MCJ expression in the liver as determined by Western blot analysis. Each lane of the blot represents an individual mouse. FIG. 9B shows a graph of ALT levels determined in serum (n = 5). ^** represents statistical significance (p <0.01) as determined by Student's t test. FIG. 10 shows a western blot showing the reduction of MCJ in human MCF7 cells transfected with h-siMCJ. CoxIV (mitochondrial protein) and GAPDH levels were also examined as controls (vehicle).

  It has now been discovered that methods and compounds that reduce MCJ polypeptide activity are useful for treating toxic diseases and conditions. Here, reducing the activity of MCJ (DnaJC15) can reduce the toxic effects on cells, organs and organ systems, and increase mitochondrial function in the liver in the absence of MCJ polypeptide activity. I confirmed that it was seen. Here, studies in a mouse model of cirrhosis (bile duct ligation, BDL) in MCJ knockout mice have demonstrated that MCJ deficiency protects against the development of fibrosis. Using a mouse model for drug-induced liver injury (DILI) involving administration of high doses of acetaminophen, MCJ polypeptide knockout mice have been shown to be more resistant to DILI than wild type mice. It was. Furthermore, here it has been confirmed that treatment with siRNA for MCJ protects wild-type cells from toxic injury, for example, but not intended to be limiting, administration of siRNA for MCJ is caused by acetaminophen. It was found to protect wild type hepatocytes from the onset of toxic injury.

  Methylation-regulated J protein (MCJ) / DnaJC15 is a member of the DnaJC subfamily of cochaperones. MCJ is a small protein of 150 amino acids and is a unique member of the DnaJC family. It contains a J domain located at the C-terminus, in contrast to the general N-terminal position, whose N-terminal region has no homology with any other known protein. In addition, MCJ also contains a transmembrane domain, while most DnaJ proteins are soluble. Phylogenetic studies have shown that MCJ exists only in vertebrates, where it is highly conserved (Hatle et al., 2007). The amino acid sequence of the human DNAJ domain-containing protein MCJ with GENBANK® accession number AAD38506.1 is set forth herein as SEQ ID NO: 1. SEQ ID NO: 2 is the mRNA sequence of human DNAJ domain-containing protein MCJ described as GENBANK (registered trademark) accession number AF126743.1. GENBANK® accession number AAH95400.1 provides the amino acid sequence of the subfamily C human DnaJ (Hsp40) homolog, which is provided herein as SEQ ID NO: 4. SEQ ID NO: 5 is the nucleotide sequence of the subfamily C human DnaJ (HSP40) homolog described as GENBANK® accession number BC095400.1. SEQ ID NO: 6 is the human MCJ cDNA sequence.

  Here, the level of cellular MCJ polypeptide activity correlates with the presence or absence of a drug-borne disease or condition, and a reduced level of MCJ polypeptide activity is associated with ingestion of an induced drug or agent It has been determined that this represents a reduction in the risk of a subject having a drug-borne disease or condition. Those skilled in the art will appreciate that terms such as higher, lower, reduced, reduced, increased, etc. may represent relative levels or values as compared to control levels or values.

  The present invention, in part, reduces the activity (eg, level and / or function) of MCJ / DnaJC15 (also referred to herein as MCJ polypeptide) to provide cells, tissues, organs, and subjects. Relates to methods for reducing toxic and drug diseases and conditions. The compositions, compounds, and methods of the present invention may be characterized by the presence of a drug or agent that causes one or more of toxicity and damage to one or more of cells, tissues, organs, and organ systems in a subject. It can be used to treat a subject having or at risk of having a sexually transmitted disease or condition. Accordingly, the methods and compounds of the present invention are useful for treating conditions in cells, tissues, and organs associated with drug toxicities in a subject. The present invention is also effective, in part, for reducing or eliminating MCJ polypeptide activity from the initial level of activity in a subject, or for treating a symptom of a drug-related disease or condition, or for treating a drug-related disease or condition. Is related to reducing / inhibiting to lower activity levels.

  In certain embodiments of the invention, modulating MCJ polypeptide activity comprises reducing MCJ polypeptide function in a cell, tissue, and / or subject. Reducing MCJ polypeptide function may be due to a decrease in the amount of MCJ polypeptide and / or a decrease in activity of MCJ polypeptide in a cell, tissue, or subject. In some embodiments, it will be appreciated that the methods of the invention reduce the activity of an MCJ polypeptide without altering the amount of MCJ polypeptide in a cell or tissue. A non-limiting example of a method of the invention for reducing the activity of an MCJ polypeptide is to contact the MCJ polypeptide with an MCJ inhibitory antibody or functional fragment thereof that binds to the MCJ polypeptide and reduces its activity. including. It will be appreciated that in some embodiments, the methods of the invention reduce the amount of MCJ polypeptide in a cell or tissue, thereby reducing MCJ polypeptide activity in the cell, tissue or subject. Non-limiting examples of methods for reducing the amount of MCJ polypeptide in a cell or tissue include reducing the amount of MCJ polypeptide in the cell and / or tissue while simultaneously reducing the amount of MCJ polypeptide in the cell and / or tissue. Contacting with a small molecule inhibitor, such as an RNA inhibitor of MCJ molecules that reduces the amount of peptide activity.

  The therapeutic methods of the invention involve administering one or more MCJ inhibitory compounds to a cell, tissue, or subject to reduce the activity of an endogenous MCJ polypeptide in the cell, tissue, and / or subject. Reducing the amount of sex MCJ polypeptide and / or reducing both the amount and activity of endogenous MCJ polypeptide.

  Molecules and compounds that inhibit MCJ polypeptide function and / or reduce MCJ polypeptide levels are referred to herein as MCJ regulatory molecules and compounds. MCJ modulatory molecules or compounds that reduce or reduce MCJ polypeptide activity are also referred to herein as MCJ inhibitory molecules or compounds. As used herein, the term “molecule” as used with respect to MCJ regulatory molecules refers to mutant MCJ polypeptides or fragments thereof, small molecule MCJ inhibitors, RNA interference molecules, antibodies or fragments thereof, MCJ polynucleotides. Point to. As used herein, the term “compound” as used with respect to an MCJ modulating compound refers to a mutant MCJ polypeptide or fragment thereof comprising additional elements such as a labeling agent, targeting agent, delivery agent, sequence tag, etc. Small molecule MCJ inhibitor, RNA interference molecule, antibody or fragment thereof, MCJ polynucleotide. Thus, in some embodiments of the invention, the MCJ modulating compound is composed of MCJ modulating molecules, and in certain embodiments of the invention, the MCJ modulating compound comprises an MCJ modulating molecule and one or more additional elements. including.

  The present invention provides a method of administering to a cell, tissue or subject a MCJ inhibitor molecule or compound in an amount effective to reduce MCJ polypeptide activity in the cell, tissue or subject as a treatment for a drug-borne disease or condition. Including. As used herein, “drug disease or condition” means a disease or condition resulting from ingestion by an subject of an agent (“drug”) that causes organ damage and injury to the subject. Examples of drug-induced diseases and conditions that can be treated by the methods of the present invention include drug-induced liver disease or condition, drug-induced renal disease or condition, drug-induced heart disease or condition, or drug-related cardiovascular disease or condition. Examples include, but are not limited to, cirrhosis, liver fibrosis, venous obstructive liver disease, idiosyncratic toxicity, Budd-Chiari syndrome, liver injury, kidney injury, drug allergy, acute kidney injury (AKI), Examples include, but are not limited to, fulminant hepatitis, cholestasis, cardiotoxicity, and alcohol consumption.

Inducing Agents for Drug-Disease Diseases and Conditions A drug-related disease or condition, when present in a subject, results from ingestion of an agent that results in the drug-related disease or condition in the subject. Examples of types of agents that can result in drug-induced diseases or conditions when ingested include, but are not limited to, ethanol, pharmaceuticals, and biological agents (non-limiting examples thereof include Herbal extracts, herbal teas, herbal dietary supplements, etc.). The means of entry of an agent that induces a drug-borne disease or condition into a subject can vary. An agent that results in a drug-induced disease or condition may be administered to a subject under a variety of circumstances, and non-limiting examples thereof are as part of a clinical treatment determined at least in part by a healthcare professional. Administration to a subject; as part of a treatment determined by the subject; and as part of a treatment determined by the subject together with the health care professional. An agent that results in a drug-borne disease or condition as described herein can be administered by a healthcare professional or can be administered by a subject.

  In some embodiments of the invention, a pharmaceutical or biological agent may be administered to a subject to prevent or treat a clinical condition or disease, and the agent is secondary to a drug-borne disease or condition. May be induced. In such examples, the pharmaceutical or biological agent may be administered to the subject for imaging, to treat the condition, or for another physiological reason, and the administered agent is also dependent on the agent. Causes side effects in subjects who are induced drug-borne diseases or conditions. For example, although not intended to be limiting, acetaminophen may be administered to a subject to reduce post-operative pain and may be effective in reducing pain, In addition, drug-related diseases or conditions such as those described herein such as acetaminophen-induced liver injury, liver failure and the like can be induced.

  As used herein, “dose” as used with respect to an inducer means the amount of agent per subject body weight (eg, in mg / kg, etc.). The dose of the inducer can be expressed as a single dose value or as a cumulative value of the amount of agent per subject weight over two or more doses of the agent to the subject. The dose of inducer administered to a subject can be determined using routine procedures that can take into account factors such as the age, weight, timing, agent clearance and additional conditions of the subject. In certain embodiments of the invention, the drug-borne disease or condition is administered to a subject at or above a threshold dose of a pharmaceutical and / or biological agent that induces the drug-borne disease or condition. Can be attributed to The threshold dose of inducer is a predetermined amount of agent per subject body weight administered to a subject (e.g., mg), wherein the drug-induced disease or condition is the dose induced by the agent at that dose or above. / Kg). A threshold dose of an inducing agent can refer to a single or cumulative amount of an agent that induces a drug-borne disease or condition. For example, without intending to be limiting, a pharmaceutical and / or biological agent may result in a drug-borne disease or condition when administered to a subject at or above a threshold dose for the agent And when administered to a subject at a dose below a threshold level cannot result in a drug-borne disease or condition. In some embodiments of the invention, the threshold dose of the agent may be essentially the same in 2, 3, 4, 5, 6 or more subjects, in most or all of the subjects. May be essentially the same in the subject.

  In certain aspects of the invention, the drug-borne disease or condition may result from administration or ingestion of an agent that produces a idiosyncratic response in the subject. In certain embodiments of the invention, the idiosyncratic response results in an administered pharmaceutical or biological agent that induces a drug-induced disease or condition in the subject, but not in a subject lacking characteristics. It can be the result of one or more specific physiological, medical, genetic, and / or other characteristics of an individual subject. For example, without intending to be limiting, a dose of contrast agent can result in a drug-induced disease or condition when administered to one subject, but is the same as that administered to a second subject, or multiple subjects The relative dose does not result in a drug-borne disease or condition in the second subject or subjects. In another non-limiting example, the dose of an herbal extract, such as ephedula or birch hippo, is administered to the subject (or to the first subject or subjects without causing the drug-related disease or condition described herein) Equivalent doses per subject weight of the same herbal extract taken by the second subject can result in a drug-borne disease or condition in the second subject. In some aspects of the invention, the drug-related disease or condition described herein is a subject that has ingested or was administered a pharmaceutical and / or biological agent that induces the drug-related disease or condition. Due to one or more of an idiosyncratic or immunoallergic pathogen. In some embodiments of the invention, administering a pharmaceutical agent or biological agent to a subject is not a basis for administering a pharmaceutical agent or biological agent to the subject, Or because of a physiological condition present in a subject that may be absent in all other subjects, may result in idiosyncratic or immunoallergic pathogens induced in the subject.

  Delivery means by which agents that induce drug-induced diseases or conditions, such as those described herein, can enter a subject include ingestion, as defined herein, by inhalation, injection, absorption , Infusion, infusion, ingestion, drinking, feeding, and other means. Non-limiting examples of delivery by absorption include, but are not limited to, transdermal revenue, absorption across the mucosa, absorption by cracks in the skin or membrane, absorption to the eye, and the like.

  Numerous agents that, when present in a subject, can result in drug-induced diseases or conditions that can be treated by methods and / or compounds that reduce MCJ polypeptide activity. Non-limiting examples of pharmaceuticals that can result in drug-induced diseases or conditions when present in a subject include statins, antidepressants, antibiotics, benzodiazepines, nicotinic acid, tacrine, aspirin, quinidine, NSAIDs (aspirin, indomethacin, ibuprofen , Naproxen, piroxicam, nabumetone), acetaminophen, phenytoin, isoniazid, diclofenac, augmentin (amoxicillin / clavulanic acid combination), minocycline, nitrofurantoin, fenofibrate, methamphetamine, amphetamine, Erythromycin, chlorpromazine, cotrimoxazole (combination of sulfamethoxazole and trimethoprim), amitriptyline, temazepam, diazepam, potassium Bamazepine, ampicillin, rifampin, estradiol, captopril, oral contraceptive pill (oral contraceptive), anabolic steroid, disulfiram, vitamin A, haloperidol, imipramine, tetracycline, phenytoin, methotrexate, amiodarone, methyldopa, chemotherapeutic agent, contrast agent, thiazine (Including but not limited to phenothiazine), chloramphenicol, digoxin, digitoxin, oxazepam, phenobarbital, quinidine, vancomycin, theophylline, verapamil, interferon (including but not limited to interferon beta 1a), and Warfarin is mentioned.

  Non-limiting examples of biological agents that, when present in a subject, can result in a drug-borne disease or condition include herbal agents and extracts. Examples of herbal extracts that can result in drug-borne diseases or conditions as described herein include ephedula, birch, chaparral, valerian, cypress extract, birch extract, and birch leaves, It is not limited to.

  Presence in a subject of drug-related diseases and conditions resulting from ingestion of pharmaceuticals and / or biological agents, blood tests, scans, urinalysis, etc. used in the art to assess organ function in the subject Can be identified using routine diagnostic methods such as:

Subjects and Controls In some aspects of the invention, the subject is a human or vertebrate mammal including but not limited to dogs, cats, horses, cows, goats and primates, such as monkeys. Thus, the present invention can be used to treat diseases or conditions in human and non-human subjects. In some aspects of the invention, the subject may be an agricultural animal, zoo animal, domestic animal or non-domestic animal, and the method of the invention may be used in a veterinary prevention and treatment regimen. In some embodiments of the invention, the subject is a human and the methods of the invention can be used in human prevention and treatment regimens.

  Non-limiting examples of subjects to which the invention can be applied include those diagnosed with or suspected of having a drug-borne disease or condition, or A subject that has a risk of having. The methods of the invention can be applied to subjects who have been diagnosed with a drug-borne disease or condition or who have or are at risk of developing a drug-borne disease or condition at the time of treatment. In some aspects of the invention, the drug-induced disease or condition is an acute drug-related disease or condition, and in certain embodiments of the invention the drug-related disease or condition is a chronic drug-related disease or condition.

  In some embodiments of the invention, the subject has the following metabolic disease or condition: overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia Disease, steatosis (eg, metabolic hepatic steatosis, cardiac steatosis, renal steatosis, muscle steatosis), abetalipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; hepatitis, hepatitis , Steatohepatitis, hepatitis C, genotype 3 hepatitis C, alpha 1-antitrypsin deficiency, acute pregnant fatty liver, and Wilson disease; metabolic kidney disease; hypertension, ischemia, heart failure Metabolic heart disease such as cardiomyopathy; poisoning; HIV; metabolic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease; one or more of motor and cancer.

  In some embodiments of the invention, the subject has the following metabolic disease or condition: overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia Disease, steatosis (eg, metabolic hepatic steatosis, cardiac steatosis, renal steatosis, muscle steatosis), abetalipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; hepatitis, hepatitis , Steatohepatitis, hepatitis C, genotype 3 hepatitis C, alpha 1-antitrypsin deficiency, acute pregnant fatty liver, and Wilson disease, etc .; metabolic kidney disease; hypertension, ischemia, heart failure Metabolic heart disease such as cardiomyopathy; poisoning; HIV; metabolic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease; and / or having one or more of exercise and cancer It was.

  MCJ polypeptide activity (eg, MCJ polypeptide level and / or MCJ polypeptide function) can be determined according to the present invention and compared to a control value for MCJ polypeptide activity. The control may be a default value, which can take a variety of forms. It can be a single cut-off value such as the median or average value. It can be established based on comparative groups, such as groups with normal levels of MCJ polypeptide activity and groups with reduced levels of MCJ polypeptide activity. Another example of a comparison group may be a group having one or more symptoms or diagnosis of a drug-borne disease or condition and a group not having one or more symptoms or diagnosis of a drug-related disease or condition. In general, controls can be based on apparently healthy normal individuals or apparently healthy cells in the appropriate age group.

  It will be appreciated that a control according to the present invention may be a sample of a material that is tested in parallel with the experimental material, in addition to a predetermined value. Examples include a sample from a control population or a control sample produced by manufacturing to be tested in parallel with an experimental sample.

  In some aspects of the invention, the value of MCJ polypeptide activity determined for a subject may serve as a reference value for later determination of MCJ polypeptide activity in that same subject, and thus “ Allows assessment of changes from "baseline" MCJ polypeptide activity. Thus, an initial MCJ polypeptide activity level may be present and / or determined in a subject, and the methods and compounds of the invention can be used to reduce the level of MCJ polypeptide activity in a subject. The initial level serves as a control level for the subject. Using the methods and compounds of the invention, MCJ polypeptide activity in a subject is at least 0.5%, 1%, 5%, 10% as compared to an initial level as a treatment for a drug-borne disease or condition in the subject. 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

Treatment In certain embodiments of the invention, the MCJ inhibitor compound is administered to a subject when one or more of the agent that induces a drug-induced disease or condition is before, during and after ingestion by the subject. obtain. For example, the subject is at least administered one or more pharmaceuticals and / or biological agents known or suspected of having the potential to cause a drug-borne disease or condition A subject can be pretreated with an administration of an MCJ-modulating compound that inhibits MCJ activity in the subject's cells if they are to receive clinical treatment. In some embodiments of the invention, an MCJ inhibitor compound is combined with an agent known or suspected of causing a drug-induced disease or condition in a subject. Can be administered. Under certain circumstances, the subject has taken or has taken an agent known or suspected of having the potential to induce a drug-induced disease or condition After being identified as suspected, the subject can be administered an MCJ inhibitor compound.

  In some aspects of the invention, the subject has or is at risk of developing a drug-borne disease or condition. A subject at risk of developing a drug-borne disease or condition is a subject who has an increased likelihood of developing a drug-related disease or condition as compared to a control risk of developing the drug-borne disease or condition. In some embodiments of the present invention, the level of risk may be statistically significant compared to the risk control level. A subject at risk is, for example, treated, scheduled to be treated, or treated with a dose of a pharmaceutical known or suspected of causing a drug-borne disease or condition Subjects who have taken a biological agent known or suspected of causing a drug-induced disease or condition; rather than control subjects who do not have an existing disease or genetic abnormality Subjects with pre-existing diseases and / or genetic abnormalities that make the subjects more susceptible to drug-induced diseases or conditions; subjects with a family history of drug-related diseases or conditions and / or individual history; induce drug-related diseases or conditions An agent suspected of taking an agent such as a medicinal product, biological agent, etc. Mention may be subject sometimes treated so far with respect to state. Existing diseases and / or genetic abnormalities that make a subject more susceptible to a drug-borne disease or condition have previously been identified as correlated with a higher likelihood of developing the drug-induced disease or condition It is understood that there may be a certain disease or genetic abnormality. In certain aspects of the invention, a drug-borne disease or condition as described herein may have one or more of idiopathic or immunoallergenic pathogenic basis.

  As used herein, the terms “treat”, “treated” or “treating” when used in reference to a drug-induced disease or condition are subjects that develop the drug-related disease or condition. To prevent the drug disease or condition from becoming more progressive (eg, more severe) to prevent or reduce the level of the drug disease or condition. And / or the subject has developed a drug-borne disease or condition to slow the progression of the drug-borne disease or condition in the subject as compared to a subject in the absence of therapy to reduce MCJ activity Can refer to later treatment.

  The present invention, in some aspects, relates to a method of modulating MCJ polypeptide activity in a cell, tissue, and / or subject. As used herein, the term “modulating” means altering the level of MCJ polypeptide activity (eg, MCJ polypeptide level and / or function) in a cell. In some embodiments of the invention, altering MCJ polypeptide activity comprises altering the level of MCJ polypeptide in a cell or tissue. Thus, reducing the activity of MCJ polypeptide in a cell includes reducing the level (eg, amount) of MCJ polypeptide in the cell. Accordingly, some embodiments of the present invention provide MCJ inhibition in an amount effective to reduce MCJ polypeptide activity in a cell, tissue or subject as a treatment for a drug-induced disease or condition. It includes a method of administering the compound. Cirrhosis, liver fibrosis, venous obstructive liver disease, idiosyncratic toxicity, Budd-Chiari syndrome, liver injury, kidney injury, drug allergy, acute kidney injury (AKI), fulminant hepatitis, cholestasis, cardiotoxicity, and Drug-induced diseases and conditions such as alcohol consumption may be treated by administering an MCJ inhibitor, thereby treating the subject with decreased MCJ polypeptide activity in the cell, tissue or subject.

  A variety of MCJ modulating compounds, including MCJ inhibitory compounds, can be used in the methods of the invention. As an example of an MCJ inhibitor compound that reduces the amount and / or activity of an MCJ polypeptide, in some embodiments of the invention, an MCJ molecule that may be a mutant MCJ or a polynucleotide encoding a mutant MCJ polypeptide; Peptide antibodies or functional fragments thereof, and small molecule MCJ inhibitors. In certain aspects of the invention, a compound useful for treating a drug-borne disease or condition is an MCJ polypeptide or variant that interferes with and thereby reduces the activity of a wild-type endogenous MCJ polypeptide. A polynucleotide encoding an MCJ polypeptide. In some aspects of the invention, compounds useful for treating drug-induced diseases or conditions are antibodies or antibodies that bind to MCJ polypeptides or other cellular components to reduce the activity and function of MCJ polypeptides. Including functional fragments thereof. In some aspects of the invention, the small molecule MCJ inhibitor is a small interfering RNA (siRNA) molecule, small hairpin RNA (shRNA) molecule, antisense DNA oligo, small guide RNA (sgRNA) molecule, or transcriptional activation. A factor-like effector nuclease (talen) molecule. SiRNA molecules for MCJ are also referred to herein as siMCJ oligos. siRNA activates RNAi and causes degradation of mRNA in a sequence-specific manner that relies on complementary binding of the target mRNA, a double-stranded RNA oligonucleotide with a two nucleotide 3 'terminal overhang ( Oligo). It is understood that the siRNA molecule is a double stranded RNA oligo and presents the siRNA sequences contained herein that are single stranded, in accordance with standard practice in the art. Small hairpin RNA (shRNA) contains a loop structure and, when delivered to a cell (eg, but not intended to be limiting, as part of a plasmid), it is processed into siRNA in the cell and Cause degradation of mRNA in a sequence-specific manner that relies on complementary binding of the target mRNA. Methods for designing and using RNA interference molecules are known in the art, see, eg, J. Org. K. Jung & J. D. Sander 2013, Nature Reviews Molecular Cell Biology, 14, 49-55; Ma, Y. et al. Et al., 2014 FEBS J. et al. Dec; 281 (23): 5186-93; Peng, J .; FEBS J. et al. March 3, 2015. doi: 10.1111 / febs. 13251; Niguita, G .; Et al., 2015 Front Bioeng Biotechnol. May 25, Vol 3, Article 37. doi: 10.3389 / fbioe. 2015.00037; and Lagana, A .; Methods Mol Biol 2015; 1269: 393-412; Methods Mol Biol 2014; 1097: 477-90. doi: 10.1007 / 978-1-62703-709-9 — 22; Adv Drug Deliv Rev February 7, 2015. pii: S0169-409X (15) 00009-5. doi: 10.016 / j. addr. 2015.10.07; and Methods Mol Biol. 2015; 1218: 1-15. doi: 10.1007 / 978-1-4939-1538-5_1. Please refer to.

  Non-limiting examples of polynucleotide sequences that can be used in the RNA interference methods of the present invention are described herein as SEQ ID NO: 7 and SEQ ID NO: 21. Additional sequences useful in some embodiments of the RNA interference methods of the present invention are also provided and are described herein as SEQ ID NO: 8-SEQ ID NO: 20 and SEQ ID NO: 22. Additional sequences useful in the RNA interference methods of the present invention can be prepared using design criteria to identify sequences that inhibit human MCJ expression. Non-limiting examples of one or more design criteria that can be used to prepare sequences for use in the RNA interference methods of the present invention include (1) sequences identified (using a BLAST search (Obtain) unique to the gene of interest, (2) the sequence exceeds 100 bp from the start and end of translation, (3) the sequence has no more than 3 adenines or thymidines in a row, (4) the sequence The GC content of> 30%, and (5) an approximately 19 nt target sequence is flanked in the mRNA with AA at the 5 ′ end and in some cases TT at the 3 ′ end Is to get. Additional criteria for identifying target sequences and appropriate sequences in the use of the RNA interference methods of the present invention are routinely used in the art.

  In some embodiments, the methods of the invention directly reduce the level of MCJ polypeptide in a cell, tissue, or subject, for example, by delivering an MCJ inhibitor compound to the cell, tissue, or subject, Treating a drug-borne disease or condition may be included. To treat a drug-induced disease or condition in a subject, one or more cells may be contacted with an MCJ inhibitor compound, which results in a reduced level of MCJ polypeptide activity in the cell. An MCJ inhibitor compound can be administered to a subject when cells to be contacted with the MCJ inhibitor compound are present in the subject. Cells in which MCJ activity can be reduced using the methods and MCJ inhibitor compounds of the present invention include, but are not limited to, liver cells, kidney cells, heart cells, and circulating cells.

MCJ Inhibitory Molecules and Compounds The MCJ inhibitor compounds of the present invention can be administered to a subject in an amount and manner effective to reduce the amount and / or activity of MCJ polypeptide in the subject to treat a drug-borne disease or condition. . The methods of the invention, in some embodiments, include administering one or more MCJ inhibitor compounds to a subject in need of such treatment to reduce a drug-borne disease or condition in the subject. The MCJ inhibitor compounds of the present invention may be administered to reduce MCJ polypeptide activity in the subject's liver, kidney, heart tissue, or other cells, cells, tissues and organs.

  As used herein, the terms “protein” and “polypeptide” are used interchangeably, and thus the term polypeptide may be used to refer to a full-length protein, and It may be used to refer to fragments of long proteins. As used herein with respect to polypeptides, proteins, or fragments thereof and polynucleotides encoding such polypeptides, the term “exogenous” refers to a compound that is administered to a cell or subject, and in the cell or subject. Means that it was not a naturally occurring “endogenous” molecule. It is understood that an exogenous MCJ polypeptide or MCJ polypeptide-encoding polynucleotide can be identical to an endogenous MCJ polypeptide or MCJ polypeptide-encoding nucleic acid, respectively, in terms of its sequence, but administered to a cell or subject. Is done.

  According to some aspects of the invention, full-length MCJ polypeptides or fragments of full-length MCJ polypeptides can be administered in the methods of the invention. Such MCJ polypeptides and fragments may be mutant MCJ polypeptides, and in some aspects of the invention, reduced or zero compared to normal (eg, control) MCJ polypeptide activity. MCJ polypeptide of reduced function having a quantity activity of. When introduced into a cell, such a mutant MCJ polypeptide may compete with the endogenous MCJ, eg, for a target, thus reducing the activity of the endogenous MCJ polypeptide. Variant polypeptides and fragments thereof may be naturally occurring fragments or may be synthesized using methods known in the art and tested for function using methods known in the art. May be. See, for example, the methods described in International Patent Application No. PCT / US2013 / 049885 and US Pat. No. 8,354,237. The contents of each of which are incorporated herein by reference. Full-length variant MCJ polypeptides and fragments thereof useful in the methods and compositions of the invention may be recombinant polypeptides.

  A “variant” wild-type or mutant full-length MCJ polypeptide or fragment thereof useful in the methods of the invention comprises amino acid or non-amino acid portion deletions, point mutations, truncations, amino acid substitutions and / or additions. obtain. The modification of the polypeptide of the present invention may be made by modification of a nucleic acid sequence encoding the polypeptide, or the modification may be performed by cleavage, addition of a linker molecule, addition of a detectable moiety such as a fluorescent label, etc. Or directly to the polypeptide. Modifications also include fusion proteins that include all or part of the amino acid sequence of the polypeptide.

  A fragment of a full-length wild-type or mutant MCJ polypeptide may comprise at least a maximum of n-1 contiguous amino acids of a full-length MCJ polypeptide having a contiguous sequence found in the MCJ polypeptide or in the variant. Good ("n" equals the number of amino acids in the full length MCJ polypeptide). Thus, for example, a fragment of a 150 amino acid long MCJ polypeptide is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, Or 149 consecutive amino acids (including each integer in between).

  In general, variant MCJ polypeptides may include polypeptides that have been specifically modified to alter the characteristics of the polynucleotide associated with its bioactivity. MCJ polypeptides can be synthesized with modifications, and / or modifications can be made in MCJ polypeptides by selecting and introducing amino acid substitutions, deletions, or additions. Subsequently, the modified polypeptide is tested for one or more activities, such as reducing MCJ polypeptide activity in a cell or subject, and efficacy as a treatment for a drug-induced disease or condition, and which It can be determined whether the modification provides a variant polypeptide with the desired properties.

  In some embodiments of the invention, the level or function of the MCJ polypeptide can be reduced by genetically introducing an MCJ inhibitory compound into the cell. Targeting agents and methods may be used to facilitate delivery of MCJ inhibitor compounds to specific cell types, cell subtypes, organs, spatial regions within a subject, and subcellular regions within a cell. Methods known in the art, such as genetic targeting, can also be used in embodiments of the invention to control the amount of MCJ inhibitor compound in a cell and / or subject. Some embodiments of the invention may comprise a reagent for genetically targeted expression of an MCJ inhibitor molecule, eg, a mutant MCJ polypeptide, wherein the reagent is an MCJ mutant polypeptide or a thereof. A vector containing a nucleic acid encoding the fragment is included.

  Certain embodiments of the invention include methods of administering an antibody or antigen-binding fragment thereof that specifically binds to an MCJ polypeptide and reduces MCJ polypeptide activity. Such antibodies, or antigen-binding fragments thereof, can be administered to cells and / or subjects to inhibit MCJ polypeptide activity in the cells and / or subjects. The term “antigen-binding fragment” of an antibody as used herein refers to one of the antibodies that retains the ability to specifically bind to an antigen (eg, an MCJ polypeptide) and reduce the activity of the antigen. Refers to multiple parts. Methods and routine procedures known in the art can be used to prepare and test antigen-binding fragments of MCJ activity-inhibiting antibodies for use in the methods of the invention. In some embodiments of the invention, the antibody is a recombinant antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody or a chimeric antibody, or a mixture thereof. Examples of antibodies known to specifically bind to MCJ polypeptides include monoclonal antibody i) WN. F3; ii) WN. Produced from the hybridoma cell line N-MCJ 3C1.5A12 deposited under ATCC number # PTA-8133. A12; and iii) WN. Produced from the hybridoma cell line N-MCJ 2A2.5E4 deposited under ATCC number # PTA-8134. Include, but are not limited to, E4 (see US Pat. No. 8,354,237, the contents of which are incorporated herein by reference). WN. F3, WN. A12 and WN. The E4 antibody is an example of an antibody that can be used in the methods of the invention as an MCJ modulating compound. Additional antibodies for use in the methods of the invention can be used using methods known in the art, in conjunction with the disclosure herein and the disclosure described in US Pat. No. 8,354,237. May be produced and tested, as well as Hatle et al., Mol Cell Biol. See also April 2007; 27 (8): 2952-66. The contents of each of which are incorporated herein by reference.

  Additional MCJ modulating compounds that can be administered in the therapeutic methods of the invention include small molecules or chemicals that inhibit MCJ polypeptide activity. The examples provided herein are, without intending to be limiting, RNA interference (RNAi) molecules. RNAi molecules are administered to cells, tissues and subjects to inhibit gene expression. In RNA interference methods, RNA may be administered to cells and / or subjects, where RNA molecules bind to other specific mRNA molecules and reduce their activity, thereby reducing mRNA to protein. Reduce translations. In some embodiments of the invention, RNAi methods are used to reduce MCJ polypeptide expression in cells, tissues and / or subjects. Accordingly, the methods of the invention administer to a cell and / or subject one or more RNAi molecules in an amount effective to reduce MCJ polypeptide expression, thereby causing MCJ in the cell and / or subject. It may include reducing polypeptide activity. Methods for identifying and testing such small molecules, such as RNA interference molecules, may involve the use of library screening and experimental procedures known in the art, in conjunction with the teachings provided herein. Examples of types of RNAi molecules that can be used in the methods of the present invention to inhibit MCJ polypeptide activity include small interfering RNA (siRNA), small hairpin RNA (shRNA), antisense DNA oligo, small guide RNA (SgRNA) and transcriptional activator-like effector nuclease (talen), but are not limited to these. Methods for designing and using RNAi molecules are known in the art, see for example J. Org. K. Jung & J.J. D. Sander 2013 Nature Reviews Molecular Cell Biology 14, 49-55; Ma, Y. et al. Et al., 2014 FEBS J. et al. December; 281 (23): 5186-93; Peng, J. et al. FEBS J. et al. March 3, 2015. doi: 10.1111 / febs. 13251; Niguita, G .; Et al., 2015 Front Bioeng Biotechnol. March 25, Vol 3, Article 37. doi: 10.3389 / fbioe. Lagana, A .; Methods Mol Biol. 2015; 1269: 393-412; Methods Mol Biol 2014; 1097: 477-90. doi: 10.1007 / 978-1-62703-709-9 — 22; Adv Drug Deliv Rev February 7, 2015. pii: S0169-409X (15) 00009-5. doi: 10.016 / j. addr. 2015.10.07; and Methods Mol Biol. 2015; 1218: 1-15. doi: 10.1007 / 978-1-4939-1538-5_1.

  The MCJ polypeptide-modulating compounds of the invention can be administered alone or in combination with one or more additional compounds. An MCJ inhibitor compound administered to a subject or cell to treat a drug-induced disease or condition acts in a synergistic manner with one or more other therapeutic agents or treatments. Or it may increase the effectiveness of the activity and / or increase the effectiveness of the MCJ inhibitor compound in treating a drug-borne disease or condition.

  The treatment methods of the present invention comprising administration of an MCJ inhibitor compound may be performed prior to the onset of a drug-induced disease or condition and / or early, mid- and late stages of the drug-related disease or condition and all before and after any of these stages. It can be used when a drug-related disease or condition exists, including time. The methods of the present invention also include one or more other previously unsuccessful treatments of drug-induced diseases or conditions in a subject, minimal success, and / or no longer likely to succeed. In some cases, the subject will be treated with a medication that has been treated for a drug-related disease or condition.

  It is understood that additional MCJ modulating compounds can be identified and used in the methods of the invention. For example, candidate compounds can use their assays and methods presented herein to their ability to reduce MCJ polypeptide activity (level and / or function) and their ability to treat drug-borne diseases or conditions. Can be tested.

Components of MCJ Inhibitory Compound MCJ-modulating compounds of the present invention described herein (compounds containing mutant MCJ molecules, anti-MCJ polypeptide antibodies or functional fragments thereof, small molecule MCJ inhibitors, etc.) Can be administered alone or in combination with other components such as targeting agents, labeling agents, delivery agents that cross membranes, and the like. Thus, in some embodiments of the invention, the MCJ inhibitor compound comprises an MCJ inhibitor molecule and optionally one or more additional components.

  Useful targeting agents according to some embodiments of the methods of the present invention include MCJ inhibitor compounds of the present invention in cells to be treated, such as liver cells, heart cells, circulating cells, kidney cells, hepatocytes, and the like. An agent that induces into the cell to be treated may be included. The targeting compound chosen will depend on the nature of the drug-borne disease or condition. In a non-limiting example, in some embodiments, it may be desirable to target the MCJ inhibitory compound to liver cells, kidney cells, etc. and / or liver cells, kidney cells, etc. It will be appreciated that in some embodiments of the methods of the invention, the MCJ inhibitor compound comprises exactly the MCJ inhibitor molecule, without any additional binding molecules. For example, in some aspects of the invention, an RNAi molecule can be administered to a cell and / or subject in a “naked” form, meaning that there is no delivery molecule, label, etc. attached to the RNAi molecule.

  If an MCJ inhibitor molecule is bound to or present in a composition with one or more cell or tissue-carrier agents, targeting agents, labeling agents, delivery agents, etc., one of ordinary skill in the art Recognizing suitable agents for use in the method, it is possible to select and use them. In some aspects of the invention, the carrier agent comprises one or more of nanocarriers, cell permeable peptides, polymers, dendrimers, siRNA bioconjugates, and lipid-based siRNA carriers.

  In some aspects of the invention, the targeting agent may be a mitochondrial targeting agent that is part of the MCJ inhibitor compound administered in the methods of the invention. Delivery agents for RNAi molecules are well known in the art and include, but are not limited to, aptamers; galactosamine; NAc galactosamine; PEG; cholesterol; lipids; cationic cell permeable peptides; However, it is not limited to these. Gramicidin S-based mitochondrial targeting agent, carnitine-acylcarnitine translocase system, cytochrome, malate dehydrogenase are utilized as targeting agents that can be used to deliver MCJ inhibitor molecules and compounds of the present invention to mitochondria Examples include, but are not limited to, active substances. Examples of targeting agents that can be used in some embodiments of the present invention are described by Diekert, K. et al. Et al., PNAS (1997) vol 96, no. 21, pp. 11752-11757; Addya, S .; J. et al. Cell Biology, (1997) Vol. 139, no. 3, 589-599; Del Gaizo, V .; Et al. (2003) Mol. Gen. and Metabol. Vol 80, pages 170-180, but are not limited thereto. The contents of each of which are incorporated herein by reference. Further delivery and targeting means and procedures known in the art are described in Ther Deliv., Which discloses cationic cell penetrating peptides as vehicles. 2015 Apr; 6 (4): 491-507. doi: 10.4155 / tde. 15.2; Int J Mol Sci., Describing various delivery methods, including the use of naked siRNA for inhalation delivery. March 6, 2015; 16 (3): 5254-70. doi: 10.3390 / ijms1603525; Nanomedicine (Londo), which discloses methods and materials related to the use of nanoparticles as a delivery system for siRNA. 2015 Apr; 10 (7): 1165-88. doi: 10.2217 / nnm. 14.214; Methods Mol Biol. Disclosing methods for targeting selected cells (eg, using conjugation to peptides, antibodies, etc.). 2015; 1218: 201-16. doi: 10.1007 / 978-1-4939-1538-5 — 12; Bioengineered., which describes the use of copolymers for delivery (also referred to as polyplexes). May-June 2014; 5 (3): 152-4. doi: 10.161 / bioe. 288062. Epub Feb 3, 2014; Curr Pharm Biotechnol. Describes the use of liposomes, nanoparticles and lipid nanoparticles for delivery. 2014; 15 (7): 659-72; and Adv Drug Deliv Rev., which describes the use of liposomes and nanoparticles for delivery means. February 2014; 66: 110-6. doi: 10.016 / j. addr. 2013.12.2.08. It is described on December 30, 2013. The contents of each of which are incorporated herein by reference.

  Labeling agents may be used in the methods of the invention to determine the location of MCJ polypeptides in cells and tissues, and to assess the location of cells, tissues, or organelles of an administered therapeutic compound. May be used. Procedures for binding and utilizing labeling agents such as enzyme labels, dyes, radiolabels and the like are well known in the art.

Effective amount of therapeutic method MCJ-modulating compound of the present invention (including anti-MCJ antibody or functional fragment thereof, mutant MCJ polypeptide-encoding polynucleotide, mutant MCJ polypeptide, small MCJ inhibitor molecule, etc.) A subject is administered in an amount effective to treat a drug-induced disease or condition. An “effective amount to treat a drug-borne disease or condition” is an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a compound of the invention (i) slows or stops the progression of a disease or condition, or (ii) reverses or reduces one or more symptoms of a drug-borne disease or condition Or it may be the amount necessary to eliminate. In some aspects of the invention, an effective amount when administered to a subject in need of treatment of a drug-induced disease or condition results in MCJ inhibition that results in a therapeutic response that prevents and / or treats the drug-related disease or condition. The amount of the compound. According to some aspects of the invention, an effective amount is combined with another therapeutic treatment for a drug-induced disease or condition or, when co-administered, a treatment that prevents and / or treats the drug-related disease or condition. The amount of MCJ inhibitory compound that produces a positive response. In some embodiments of the invention, the biological effect of treating a subject with an MCJ inhibitor compound can be amelioration and / or absolute elimination of symptoms due to a drug-borne disease or condition. In some embodiments of the invention, the biological effect is a complete suppression of the drug-borne disease or condition, as is apparent from, for example, a diagnostic test showing that the subject does not have the drug-borne disease or condition. It is.

  Usually, an amount of MCJ agonist compound effective to reduce MCJ polypeptide activity is determined in a clinical trial and establishes an effective dose relative to the test population relative to the control population in a blinded trial. In some embodiments, an effective amount is an effective amount that produces a desired response, eg, an amount that reduces a drug-related disease or condition in cells, tissues, and / or subjects with the drug-related disease or condition. Thus, an amount effective to treat a drug-borne disease or condition that can be treated by reducing MCJ polypeptide activity, when administered, is the amount of MCJ polypeptide activity in the subject, without administration of MCJ inhibitor molecules. In an amount that is reduced to an amount less than that present in the cell, tissue, and / or subject. In certain aspects of the invention, the level of MCJ activity present in a cell, tissue, and / or subject that has not been contacted with or administered MCJ inhibitory compounds is referred to as a “control” amount. When treating a drug-borne disease or condition, the desired response may be when reducing or eliminating one or more symptoms of the drug-borne disease or condition in a cell, tissue, and / or subject. The reduction or elimination may be temporary or permanent. It is understood that the status of a drug-borne disease or condition can be monitored using methods to determine MCJ polypeptide activity, symptom assessment, clinical trials, and the like. In some aspects of the invention, the desired response to treatment of a drug-induced disease or condition can also delay the onset of the drug-related disease or condition, or even prevent the onset of the drug-related disease or condition. can do.

  An effective amount of a compound that reduces MCJ polypeptide activity can also be determined by assessing the physiological effect of administration of the compound on a cell or subject, such as a reduction in drug-induced disease or condition after administration. Subject assays and / or symptom monitoring can be used to determine the effectiveness of the pharmaceutical compounds of the invention to determine the presence or absence of a response to therapy. The examples are not intended to be limiting, but use of tests known in the art of liver function to determine the status of a drug-induced disease or condition in a subject before and after treatment of the subject with an MCJ inhibitor compound It is. It is understood that the amount of MCJ inhibitor compound administered to a subject can be modified based at least in part on such determination of the status of the disease and condition. The amount of treatment is, for example, by increasing or decreasing the amount of MCJ inhibitor compound, changing the composition of the MCJ inhibitor compound administered, changing the route of administration, changing the timing of dosing, etc. Can be changed. An effective amount of an MCJ inhibitor compound depends on the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the combination therapy (if any), It varies according to the specific pathway and further factors within the knowledge and views of medical personnel. For example, an effective amount can depend on the desired level of MCJ polypeptide activity that is effective in treating a drug-induced disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular MCJ inhibitor compound of the present invention without necessitating undue experimentation. In combination with the teachings provided herein, a variety of MCJ inhibitor compounds can be selected to determine efficacy, relative bioavailability, patient weight, severity of adverse side effects, preferred mode of administration, etc. By weighing the factors, an effective prophylactic or therapeutic treatment regimen that is effective in treating a particular subject can be planned.

  MCJ inhibitor compounds administered using the methods of the invention are also referred to herein as “pharmaceutical compounds”. The pharmaceutical compound dosage may be adjusted by an individual medical professional or veterinarian, especially in the case of any complication. A therapeutically effective amount is typically 0.01 mg / kg to about 1000 mg / kg, about 0.1 mg / kg to about 200 mg / kg for one or more days, administered once or multiple daily doses, or It varies from about 0.2 mg / kg to about 20 mg / kg. The absolute amount will depend on a variety of factors, including combination treatment, number of doses and individual subject parameters including age, physical condition, size and weight. These are factors well known to those skilled in the art and can be addressed using only routine experimentation. In some embodiments, the maximum dose, ie the highest safe dose according to sound medical judgment, can be used.

  The methods of the invention include, in some cases, administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of the MCJ inhibitor compound. In some cases, a pharmaceutical compound of the invention (eg, an anti-MCJ antibody or functional fragment thereof, a mutant MCJ polypeptide-encoding polynucleotide, a mutant MCJ polypeptide, a small molecule MCJ inhibitor such as an RNA interference molecule, etc.) Can be administered to a subject at least every other day, once a week, every other week, once a month, etc. The dose may be administered once per day or more than once per day, eg, 2, 3, 4, 5 or more times in one period of 24 hours. As described elsewhere herein, an MCJ inhibitor compound is a drug-related disease or drug prior to administration to a subject of an agent known to induce the drug-related disease or condition. In combination with administration of an agent known to induce a condition to a subject, or after administration to a subject of an agent known to induce a drug-induced disease or condition, or these It can be administered to a subject in any combination of times.

  The methods of the invention may be used in some subjects, alone, in combination with one or more other MCJ inhibitor compounds, and / or administered to a subject with a drug-borne disease or condition. Or administration of a pharmaceutical compound in combination with a therapeutic regimen. The pharmaceutical compound can be administered in a pharmaceutical composition. The pharmaceutical compositions used in the methods of the present invention may be sterile and have MCJ polypeptide activity at a level sufficient to produce the desired response in a unit of weight or dose suitable for administration to the subject. Containing a reducing amount of the MCJ inhibitor compound. The dosage administered to a subject of a pharmaceutical composition comprising an MCJ inhibitor compound to reduce MCJ polypeptide activity can be selected according to various parameters, particularly according to the mode of administration used and the condition of the subject. Other factors may include the desired duration of treatment. If the response in the subject is inadequate with the initial dose applied, use a higher dose (or a higher dose that is effectively higher by a different, more local delivery route) to the extent that patient tolerance permits. Also good.

Methods of Administration A variety of administration routes for MCJ inhibitor compounds are available for use in the methods of the invention. The particular delivery mode chosen will depend at least in part on the particular condition being treated and the dosage required for therapeutic efficacy. The methods of the invention generally can be any medically acceptable, meaning any manner that results in an effective level of treatment of a drug-borne disease or condition without causing clinically unacceptable adverse effects. It can be performed using a mode of administration. In some embodiments of the invention, the MCJ inhibitor compound may be administered via oral, enteral, mucosal, transdermal and / or parenteral routes. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraperitoneal and intrasternal injection or infusion techniques. Other routes include, but are not limited to, nasal (eg, via a stomach-nasal tube), dermis, intravaginal, rectal and sublingual. The delivery route of the present invention may include intrathecal, intraventricular or intracranial. In some embodiments of the invention, the compounds of the invention may be administered within a sustained release matrix, depending on the placement of the matrix in the subject. In some aspects of the invention, MCJ inhibitor compounds (such as anti-MCJ antibodies or functional fragments thereof, mutant MCJ polypeptide-encoding polynucleotides, mutant MCJ polypeptides, or small molecule MCJ inhibitors, etc.) are identified Nanoparticles coated with a delivery agent that targets cells or organelles (a non-limiting example of which is mitochondria) may be used to administer cells of interest. Various delivery means, methods, and agents are known in the art. Non-limiting examples of delivery methods and delivery agents are further provided elsewhere herein.

  In some methods of the invention, one or more MCJ inhibitor compounds may be administered in a formulation, which may be administered in a pharmaceutically acceptable solution, which is routinely Pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients may be included. In some embodiments of the invention, the MCJ inhibitor compound may be formulated with a pharmaceutical agent for co-administration. Non-limiting examples are formulations comprising MCJ inhibitor compounds and acetaminophen compounds, as well as formulations comprising MCJ inhibitor compounds and contrast agents. According to the methods of the present invention, the MCJ inhibitor composition may be administered in a pharmaceutical composition. Generally, the pharmaceutical composition comprises an MCJ inhibitor compound and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art. As used herein, a pharmaceutically acceptable carrier refers to the effectiveness of the biological activity of the active ingredient, eg, an anti-MCJ antibody or functional fragment thereof, a mutant MCJ polypeptide-encoding polynucleotide, a mutation A non-toxic material that does not interfere with the ability of a body MCJ polypeptide, or a compound such as a small molecule MCJ inhibitor, to treat a drug-borne disease or condition. Numerous methods useful for administering and delivering antibodies, polypeptides, polynucleotides, small molecules, RNAi molecules, and the like for therapeutic use are known in the art.

  Pharmaceutically acceptable carriers may include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials well known in the art. Exemplary pharmaceutically acceptable carriers are described in US Pat. No. 5,211,657, others are known to those skilled in the art. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, salts should be pharmaceutically acceptable, but pharmaceutically unacceptable salts may be used conveniently to prepare their pharmaceutically acceptable salts. And are not excluded from the scope of the present invention. Such pharmacologically and pharmacologically acceptable salts include the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid and the like. Examples include, but are not limited to, those prepared. Similarly, pharmaceutically acceptable salts can be prepared as alkali metal or alkaline earth salts, such as sodium, potassium or calcium salts.

  Some embodiments of the methods of the invention comprise administering one or more MCJ inhibitor compounds directly to the tissue. In some embodiments, the tissue to which the compound is administered is tissue that is likely to develop a drug-borne disease or condition, non-limiting examples of which are liver, kidney, heart tissue. Direct tissue administration can be achieved by direct injection or other means. Many orally delivered compounds move spontaneously to and through the liver and kidney. Some embodiments of the therapeutic methods of the invention comprise oral administration of one or more MCJ inhibitor compounds to a subject. MCJ inhibitor compounds may be administered once or in combination with other agents, or they may be administered in multiple doses. When administered multiple times, the compound can be administered via a variety of routes. For example, without intending to be limiting, the first (or first few) administration may be by oral administration, and one or more additional administrations may be oral and / or systemic administration It can be.

  For embodiments of the invention where it is desirable to administer the MCJ inhibitor compound systemically, the MCJ inhibitor compound can be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Injectable formulations may be provided in unit dosage form, eg, in ampoules, or in multiple dose containers, with or without added preservatives. MCJ inhibitor compound formulations (also referred to as pharmaceutical compositions) may take the form of suspensions, solutions or emulsions in oily or aqueous vehicles, such as suspending, stabilizing and / or dispersing agents. Of formulation agents may be included.

  Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, saline / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or non-volatile oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents and inert gases may also be present. The low dose may be due to other forms of administration, such as intravenous administration. If the response in the subject is inadequate with the initial dose applied, use a higher dose (or a higher dose that is effective by a different, more local delivery route) to the extent that patient tolerance permits. Also good. Multiple doses per day can be used as needed to achieve the appropriate systemic or local level of one or more MCJ inhibitor compounds.

  In yet other embodiments, the methods of the invention include the use of a delivery vehicle such as a biocompatible microparticle, nanoparticle, or implant that is suitable for implantation into a recipient. An exemplary biodegradable graft that is useful according to this method is described in PCT International Publication No. WO 95/24929 (incorporated herein by reference), which contains biological macromolecules. A biocompatible, biodegradable polymer matrix for inclusion is described. Such delivery means are well known in the art and can be used to achieve sustained release of MCJ inhibitor compounds in a subject and are selected to release by diffusion over a longer period of time without degradation. obtain.

  Both non-biodegradable and biodegradable polymer matrices can be used to deliver one or more MCJ inhibitor compounds to a subject in the methods of the invention. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer may be selected based on the time period for which release is desired, typically on a scale of hours to years or longer. In general, release over a period ranging from several hours to 3-12 months can be used. The polymer is optionally present in the form of a hydrogel that can absorb up to about 90% of its weight in water, and optionally further crosslinked with multivalent ions or other polymers.

  In general, MCJ inhibitor compounds may be delivered in some embodiments of the invention using biodegradable implants by diffusion or by degradation of the polymer matrix. Exemplary synthetic polymers for such use are well known in the art. Biodegradable and non-biodegradable polymers can be used for delivery of MCJ inhibitor compounds using methods known in the art. Bioadhesive polymers such as biodegradable hydrogels (see HS Sawhney, CP Pathak and JA Hubell, Macromolecules, 1993, 26, 581-587, the teachings of which are (Incorporated herein) can also be used to deliver MCJ inhibitor compounds for the treatment of drug-induced diseases or conditions. Further suitable delivery systems can include time release, delayed release or sustained release delivery systems. Such a system can avoid repeated administration of the MCJ inhibitor compound, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those skilled in the art (eg, US Pat. Nos. 5,075,109; 4,452,775; 4,675,189; No. 5,736,152; No. 3,854,480; No. 5,133,974; and No. 5,407,686, each of which is herein incorporated by reference. Incorporated)). In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

  The use of long-term sustained release grafts is being treated with a medicinal product for prophylactic treatment of the subject and at risk of developing a recurrent drug-induced disease or condition, for example, with known side effects of the drug-induced disease or condition May be appropriate for the subject. Extended release, as used herein, is constructed and arranged to deliver a therapeutic level of MCJ inhibitor compound for at least 30 days, 60 days, 90 days, or longer. Means. Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

  A therapeutic formulation of an MCJ inhibitor compound is a lyophilized formulation or an aqueous solution in which a molecule or compound having a desired degree of purity is added to any pharmaceutically acceptable carrier, excipient or stabilizer [Remington 'Pharmaceutical Pharmaceutical. Sciences 21st edition (2006)] and can be prepared for storage. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations used; buffers such as phosphate, citrate and other organic acids; ascorbic acid and methionine Preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol Low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; glycine, glu Amino acids such as min, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; A salt-forming counterion; a metal complex (eg, a Zn-protein complex); and / or a nonionic surfactant such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

Assessing therapy Assessing the effectiveness of candidate MCJ inhibitor molecules and compounds to reduce the activity of MCJ polypeptides in cells or tissues can also be used to determine candidate MCJ inhibition, for example with respect to their ability to reduce MCJ polypeptide activity. As a screening assay for evaluating compounds, it may be performed using the assay of the present invention in cells from culture. MCJ inhibitor compounds that reduce MCJ polypeptide activity in a cell, tissue, or subject are used in the treatment of a drug-borne disease or condition or as a pretreatment for a drug-borne disease or condition (eg, inducing a drug-related disease or condition Can be used to prepare cells or subjects for subsequent therapy using known agents.

  Suitable assays include determining the level of a polynucleotide encoding MCJ polypeptide and / or determining the level of MCJ polypeptide and / or MCJ polypeptide activity in a cell, tissue, and subject. , Including but not limited to, means for determining MCJ polypeptide activity. The level of MCJ polypeptide-encoding polynucleotides and polypeptides and their activity can be determined in a number of ways when carrying out the various methods of the invention. In some embodiments of the invention, the level of MCJ polypeptide-encoding polynucleotide or polypeptide or their activity is determined by the control level of MCJ polypeptide-encoding polynucleotide or polypeptide or their level in a cell, tissue, or subject, respectively. Measured in terms of activity. One possible measurement of the level of an MCJ polypeptide-encoding polynucleotide or polypeptide is a measurement of the absolute level of the MCJ polypeptide-encoding polynucleotide or polypeptide. This can be expressed, for example, in MCJ polypeptide-encoding polynucleotide or polypeptide per unit of cell or tissue. Another measure of the level of MCJ polypeptide-encoding polynucleotide or polypeptide is a measurement of the change in level and / or activity of MCJ polypeptide-encoding polynucleotide or polypeptide over time. This may be expressed in absolute amounts or in terms of percent increase or decrease over time. Activity assays for MCJ polypeptides can also be used to assess the effectiveness of MCJ inhibitor molecules or compounds. Further, in certain embodiments of the invention, antibodies or antigen-binding fragments thereof, or other compounds that specifically bind MCJ polypeptides are used to treat MCJ polypeptides present after treatment with MCJ inhibitor compounds. You can evaluate the level.

  In some embodiments of the invention, the decrease in MCJ polypeptide activity level in the cell or tissue is greater than 0.2%, greater than 0.5%, 1.0%, including all values in this range, 2.0%, 3.0%, 4.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70 %, 80%, greater than 90%, or a 100% decrease. A decrease in MCJ polypeptide activity after contact with the MCJ inhibitor compound may indicate the effectiveness of the MCJ inhibitor compound for treating a drug-induced disease or condition in the subject.

  As will be appreciated by those skilled in the art, the assessment of the treatment of the present invention may also be based on the assessment of the symptoms or clinical endpoints of a drug-borne disease or condition, such assessment being based on the status of the drug-borne disease or condition and / or Alternatively, it can be used in combination with the methods of the present invention to assess the effectiveness of treatment of a drug disease or condition. Antibodies or antigen-binding fragments or other compounds that specifically bind the MCJ polypeptide can be used to assess the level of MCJ polypeptide following treatment with an MCJ inhibitor compound. Antibodies or antigen-binding fragments or other compounds that specifically bind the MCJ polypeptide can be used to assess the level of MCJ polypeptide following administration of the MCJ inhibitor compound in the therapeutic methods of the invention.

Kits comprising MCJ inhibitor compounds and instructions for their use in the methods of the invention are also within the scope of the invention. The kit of the present invention may comprise one or more MCJ inhibitor compounds such as anti-MCJ antibodies or functional fragments thereof, mutant MCJ polypeptide-encoding nucleotides, mutant MCJ polypeptides, or small molecule MCJ inhibitors. It can be used to treat a drug-borne disease or condition. Kits containing MCJ inhibitor compounds can be prepared for use in the therapeutic methods of the invention. The components of the kit of the invention can be packaged either in an aqueous medium or in lyophilized form. The kit of the present invention may comprise a carrier that is partitioned to receive one or more container means or series of container means, such as test tubes, vials, flasks, syringes, etc., confined therein. The first container means or series of container means comprise one or more of an anti-MCJ antibody or functional fragment thereof, a mutant MCJ polypeptide-encoding polynucleotide, a mutant MCJ polypeptide, or a small molecule MCJ inhibitor, etc. A compound may be contained. The second container means or series of container means may contain targeting agents, labeling agents, delivery agents, etc. that may be included as part of the MCJ inhibitor compound administered in the therapeutic method embodiment of the invention.

  The kit of the present invention may also include instructions. The instructions are typically in written form and provide guidance for performing the treatment embodied by the kit and making decisions based on that treatment.

Methods for Identifying Candidate Compounds Certain embodiments of the invention include methods for identifying and / or screening for candidate compounds that reduce MCJ polypeptide activity in a cell, tissue, and / or subject. The method comprises contacting a candidate compound with a cell or tissue, and / or administering the candidate compound to a subject, and MCJ polypeptide before and after contacting the cell, tissue, and / or subject with the candidate compound. Determining the amount of activity may be included. A decrease in the amount of MCJ polypeptide activity compared to the appropriate control indicates a compound that is capable of reducing the level of MCJ.

  An assay mixture useful for assessing therapeutic candidates for drug-related diseases or disorders includes the candidate compound. Candidate compounds may be antibodies, small organic compounds, small molecules, polypeptides, DNA molecules, RNA molecules, etc., and thus combinatorial antibody libraries, combinatorial protein libraries, small organic molecule libraries, or any other From any suitable source. Candidate DNA or RNA molecules can be designed based on parameters recognized in the art for molecules useful for reducing gene expression. Usually, to test candidate compounds, multiple reaction mixtures are run in parallel with different compound concentrations to obtain different responses to different concentrations. Usually, one of these concentrations serves as a negative control, i.e. at zero concentration of the compound or at a compound concentration below the limit of assay detection.

  A variety of other reagents may also be included in the assay mixture to test candidate compounds. These may include reagents such as salts, buffers, neutral proteins (eg, albumin), detergents, etc., which may be used to facilitate optimal protein-protein and / or protein-compound binding. . Such reagents can also reduce non-specific or background interactions of reaction components. Other reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antibacterial agents, etc. may also be used. The order of addition of the components, incubation temperature, incubation time, and other parameters of the assay can be readily determined. Such experiments merely involve the optimization of assay parameters and do not include the basic composition of the assay. The incubation temperature is usually 4 ° C to 40 ° C. Incubation time may be minimized to facilitate rapid high-throughput screening, and is usually 0.1 to 10 hours. After incubation, variables such as the presence, amount, and / or activity of the MCJ polypeptide can be detected by any convenient method available to the user. For example, the amount and / or activity of MCJ polypeptide after contact with a candidate compound can be determined using standard methods and as described herein.

  The following examples are provided to illustrate specific cases of practicing the invention and are not intended to limit the scope of the invention. As will be apparent to those skilled in the art, the present invention applies to various compositions and methods.

Example 1
Experiments and studies were conducted to evaluate the effect of reducing the activity of MCJ (DnaJC15) on drug-induced damage and injury in cells, organs, and organ systems. The results showed that in the absence of MCJ, increased mitochondrial function in the liver was seen. Studies were conducted in a mouse model of cirrhosis (bile duct ligation, BDL) in MCJ knockout mice, and the results demonstrated that MCJ deficiency protects mice from developing fibrosis. Experiments involving mouse models for drug-induced liver injury (DILI) using high doses of acetaminophen have shown that MCJ knockout mice are more resistant to DILI than mice with normal MCJ polypeptide expression / activity Proven to be sex. In addition, experiments were performed that demonstrated that treatment with siRNA for MCJ protected wild-type cells from drug-induced (toxic) injury. The results showed that siRNA for MCJ protects wild type hepatocytes from the development of toxic injury caused by acetaminophen administration.

Materials and Methods Human Samples Liver function is preserved and has cirrhosis and hepatocellular carcinoma (HCC) corresponding to either Barcelona Clinic River Center (BCLC) stages A (n = 34) and B (n = 13) 47 Patients with Dra. Offered by Erica Villa (University of Modena Reggio Emilia, Modena, Italy). Healthy human liver samples were used as controls (n = 13). Further information is available from the original study (Villa, E. et al., 2015, Neoangiogenesis-related genes are hallmarks of fast-growing hepatocyte cellular carcinomas and worst sur. 1136 / gutjnl-2014-308483-Epub). Informed consent was obtained from all patients included in the study and was therefore accompanied by ethical principles embodied in the Declaration of Helsinki.

Animals Three month old males (C57BL6), MCJ wild type (WT) and MCJ knockout (KO) mice were used for the study. The animal procedure was approved according to the guidelines of the CIC bioGUN animal facility with AAALAC certificate.

Immunohistochemistry Paraffin-embedded liver samples were sectioned, defatted and hydrated. All procedures were performed according to standard protocols using EnVision + System HRP (Dako, Denmark). Finally, the sample was incubated with a Vector Vip substrate (Vector, USA) for color development. Images were taken from a microscope AXIO Imager A1 (Carl Zeiss AG, Germany) using a 10x or 20x objective lens. Quantification of staining intensity, average synthesis of intensity and percent of staining area for each sample are as follows: FRIDA software (Framwork for Image Data Analysis) http: // bui3. win. ad. jhu. Calculated using edu / frida /.

RNA isolation and real-time polymerase chain reaction (RT-PCR)
Total RNA was isolated using Trizol (Invitrogen, USA). 1-2 μg of total RNA was treated with DNAse (Invitrogen) and reverse transcribed into cDNA using M-MLV reverse transcriptase (Invitrogen). Subsequently, qPCR was performed using the CFX Connect ™ RT-PCR detection system (BioRad) using iQ ™ SYBR ™ Green Supermix (BioRad, USA). Expression levels were normalized to the average level of GAPDH mRNA in each sample.

Animal cirrhosis experimental model and in vivo drug treatment MCJ wild type (WT) and MCJ knockout (KO) mice bred in an animal facility at CIC bioGUNE were used. The animal procedure was approved by the CIC bioGUNE Animal Care and Use Committee. Bile duct ligation was performed as previously described (Fernandez-Alvarez S et al. (2015) Lab Invest. 95, 223-36). At least 5 animals were used per group.

Characterization of Liver Injury Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin were used in serum samples using Selecta Junior SpinLab 100 analyzer (Vital Scientific) and SpinReact reagent according to the manufacturer's protocol. Were determined.

Isolation and culture of primary hepatocytes Primary hepatocytes were isolated from male WT MCJ-KO mice by collagenase perfusion as described (Barbier-Torres L et al. (2015) Oncotarget. 6, 2509-23. See). Adherent cells were maintained in MEM with 10% fetal bovine serum (FBS).

Measurement of Oxygen Consumption Rate and Extracellular Acidification Rate Primary MCJ-WT mouse hepatocytes were each treated with 2.0 × 10 ⁴ cells per well in an XF24 cell culture microplate coated with collagen I (Seahorse Bioscience). Seeded with cells. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements were performed after 1 hour equilibration in assay medium. After OCR and ECAR baseline measurements, oligomycin (1 μM), carbonylcyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP) (3 μM) and rotenone (RO) (1 μM) solution were added sequentially to each well. The working concentration was allowed to reach and analyzed for changes in OCR and ECAR. Normalized data was expressed as O ₂ pmol per minute or millipH units per minute (mpH) per μg protein for primary hepatocytes.

Silencing in vitro Primary MCJ-WT mouse hepatocytes were transfected with 2 mg shMCJ or unrelated ShControl using Jetprim reagent (Polyplus) for 24 hours. One particular experiment involved the use of shMCJ containing the siRNA having SEQ ID NO: 22 that was included in the plasmid and delivered as part of the plasmid by standard shRNA procedures. Primary hepatocytes were treated with 100 mM deoxycholic acid (DNA) for 2 hours. Each experiment was performed using triplicates.

Apoptosis measurement The caspase 3 activity assay was performed as previously described [Barbier-Torres L et al. (2015) Oncotarget. 6, pp. 2509-23].

Statistical analysis All experiments were performed in triplicate. Data were expressed as mean ± SEM. Statistical significance was estimated using the Student t test. A p value of less than 0.05 was considered significant.

Results The results showed that MCJ levels increased in the liver in subjects with cirrhosis. 1A-1B show microscopic images and graphs showing that the livers of cirrhotic patients have increased MCJ levels. The results of immunohistochemistry in the left panel of FIG. 1A show MCJ expression in the liver from a healthy control subject (NL) (n = 5), and the results of immunohistochemistry in FIG. 1A show cirrhosis (hepatitis C). Figure 6 shows MCJ expression in livers from patients with n = 16. FIG. 1B is a graph showing the level of MCJ expression determined for healthy and cirrhotic patients. The results show a statistically significant increase in the level of MCJ in cirrhotic liver.

  The results of experiments conducted to assess the effect of the presence of MCJ on the severity of liver injury in a mouse model of drug-induced liver injury (DILI) indicate that a decrease in MCJ levels indicates that wild-type mice with normal MCJ levels It has been demonstrated to result in a reduction in the amount of drug-induced liver injury relative to the amount of drug-induced liver injury in 2A-2B provide microscopic images and graphs showing the use of acetaminophen administration as a mouse model for drug-induced liver injury (DILI). Results are shown for wild type (WT) and MCJ knockout (KO) mice that received acetaminophen intraperitoneally. FIG. 2A shows the results after 48 hours and shows the presence of macrophages in liver sections examined by immunohistochemistry for the F4 / 80 macrophage marker. 2A (left panel) represents WT and FIG. 2A (right panel) represents MCJ KO. The right graph of FIG. 2B shows the levels of ALT and AST transaminases in the serum. FIG. 2B (left panel) represents one mouse for each genotype. FIG. 2B (right panel) shows the values for n = 4 mice for each genotype. The results showed that KO mice were more resistant to damage induced by acetaminophen in the liver. FIG. 2B left panel shows fewer F4 / 80 macrophage markers in KO mice. MCJ KO mice developed less inflammation than WT mice and had lower levels of transaminase.

  Results of experiments conducted to assess the effect of the presence of MCJ on the severity of cirrhotic liver injury in a mouse model of cirrhotic liver injury indicated that a decrease in the level of MCJ was observed in the wild with normal MCJ levels. It has been demonstrated that the amount of cirrhosis-type liver damage is reduced relative to the amount of cirrhosis-type liver damage in type I mice. 3A-3C show the results from WT and MCJ KO mice that underwent biliary ligation surgery using a bile duct ligation (BDL) mouse model for cirrhosis. FIG. 3A shows a microscopic image of the liver after 14 days. Tissue damage was indicated by the presence of macrophages (F4 / 80) by IHC as a marker of inflammation. The left panel of FIG. 3A is an image derived from one WT mouse, and the right panel of FIG. 3A is an image derived from one MCJ KO mouse. FIG. 3B is a graph showing F4 / 80 positive staining values for n = 4 mice for each genotype. FIG. 3C provides survival curves for WT and MCJ KO mice during bile duct ligation surgery. MCJ KO mice are more resistant to developing cirrhotic liver damage.

  Results of experiments conducted to measure oxygen consumption rate (OCR) values in liver cells to assess whether administration of MCJ shRNA protects liver cells from cirrhotic-type damage. Administration of MCJ shRNA reduced the amount of cirrhotic damage compared to the level of cirrhotic damage in wild-type mice not receiving MCJ shRNA. 4A-4B provide graphs showing that MCJ shRNA (shMCJ) protected primary hepatocytes from DCA-induced death. FIG. 4A shows oxygen consumption rate (OCR) values in primary hepatocytes from WT mice transfected with control (WT) or shMCJ (shMCJ) expression plasmids. Results showed that shMCJ treatment reduced OCR compared to WT. FIG. 4B shows caspase-3 activity (marker for cell death) after treatment with 100 μM DCA in primary hepatocytes from WT mice transfected with control (WT) or shMCJ (shMCJ) expression plasmids. The results showed that treatment with shMCJ reduced caspase-3 activity compared to WT.

Example 2
Mitochondria are the main energy source in hepatocytes and play an important role in the liver's extensive oxidative metabolism and normal function. Mitochondria control signaling that mediates hepatocellular injury, as impaired mitochondrial function affects cell survival and contributes to liver disease. In particular, alterations in mitochondrial function have been demonstrated in various chronic liver diseases including drug-induced liver injury (DILI). The goal of the study is to determine whether MCJ, an endogenous negative regulator of mitochondrial respiration, is effective against DILI, and to inhibit drugs that can prevent or cure liver injury by inhibiting MCJ function It was development.

Materials and Methods For further methods and details, see Materials and Methods in Example 1.

Liver histology and immunohistochemistry for MCJ Paraffin-embedded liver samples were sectioned, defatted and hydrated. Immunohistochemistry (IHC) was performed using anti-MCJ antibody and nuclei were counterstained with hematoxylin. Quantification of the stained area of each sample was analyzed using Frida software and expressed as a percentage of the stained area relative to the total area (magnification field). Ten fields of view were taken and analyzed per sample. For liver histology, liver sections from paraffin-embedded tissue were stained with hematoxylin and eosin (H & E staining). Frida software was used for quantification.

Analysis in primary mouse hepatocytes Primary hepatocytes were isolated from WT MCJ KO mice by collagenase perfusion. Cells were cultured in MEM medium and treated with acetaminophen 10 mM for various time points. ATP levels were measured using the kit ATPLite (Perkin Elmer). Mitochondrial ROS was measured with MitoSOX reagent (Thermo Fisher Scientific) used for fluorescence microscopy analysis. Cell death was quantified with the TUNEL assay (Roche).

Western Blotting Analysis Analysis of MCJ expression in the liver by Western blot analysis was performed as previously described (REF) using specific anti-MCJ antibodies. GAPDH expression was used as a control. Band intensity was quantified using ImageJ software and normalized to control (GAPDH).

Intravenous (iv) administration of siRNA siMCJ oligos (double stranded RNA oligos, see eg SEQ ID NO: 22) containing the designed sequence were synthesized for in vivo studies (Ambion in vivo siRNA) . In vivofectamine 3.0 (Life Technology) was used as a delivery system because it is required to obtain high efficacy of siRNA delivery to hepatocytes in vivo. At the recommendation of the manufacturer, 1.7 mg / Kg was used in combination with in vivofectamine 3.0. Preparation of siRNA using Invivofectamine was performed as recommended by the manufacturer. Mice received a single intravenous dose of siMCJ / in vivofectamine in 150 μl.

Results Increased MCJ expression in the liver caused by drug-induced liver injury (DILI) in humans and mice Because mitochondrial function is severely impaired in the liver of patients with drug-induced liver injury (DILI), these patients and healthy MCJ expression was examined in liver biopsies obtained from various control subjects. Expression was examined by immunohistochemistry followed by further quantification (see methods). The results revealed that MCJ expression was statistically high in livers from DILI patients (FIG. 5A).

  MCJ expression in the mouse liver is also a mouse with DILI leading to severe liver damage (high transaminase, tissue damage) within 36-48 hours when mice are fed with a single high dose of acetaminophen (APAP) Inspected using model. Acetaminophen-induced liver injury is the second most common cause of acute liver failure and often requires liver transplantation as the only treatment. Analysis of MCJ expression as determined by Western blot analysis showed significantly higher levels of MCJ in livers from APAP-treated mice compared to control mice (FIG. 5B).

  Taken together, these data indicated that drug-induced liver injury caused an increase in MCJ levels in the liver in both human patients and mice. These data also suggested that disrupting MCJ expression and / function can be used as a therapeutic approach.

The lack of MCJ protects hepatocytes from acetaminophen-induced mitochondrial dysfunction and death Considering the negative role of MCJ in mitochondrial function and the increased expression of MCJ in livers with DILI, A study was conducted to investigate whether mitochondrial function could be protected in hepatocytes upon treatment with acetaminophen (APAP). Hepatocytes were isolated from the livers of WT or MCJ KO mice and treated with APAP. At a rate of 9 hours after treatment, ATP levels in WT hepatocytes were significantly lower in WT hepatocytes, but remained high in MCJ KO hepatocytes (FIG. 6A). APAP causes the production of mitochondrial ROS, which has previously been shown to be responsible for mediating hepatocyte death. Unexpectedly, the absence of MCJ in MCJ KO hepatocytes prevented the generation of ROS (FIG. 6B). Thus, the lack of MCJ is sufficient to maintain normal mitochondrial function in hepatocytes in response to APAP, preventing the generation of ROS. In correlation with these effects, MCJ KO hepatocytes were found to be more resistant to cell death caused by APAP (FIG. 6C).

These data support the conclusion that MCJ expression and / or function can be disrupted to protect the liver from drug damage.
MCJ siRNA (siMCJ) can be used to disrupt MCJ expression in the liver in vivo (pharmacodynamics)
Based on positive results from recent attempts to use siRNA to target liver molecules (REF), siRNA for MCJ (siMCJ) was used in a strategy to reduce the level of MCJ in the liver. To determine the efficacy and pharmacodynamics of the siMCJ pilot study, where wild-type mice maintained under normal conditions were administered a single intravenous dose of siMCJ in combination with in vivofectamine (as a delivery system) Subsequently, mice were collected at various times. The level of MCJ in the livers of those mice was examined by Western blot analysis. Immediately 24 hours after administration of siMCJ, the level of endogenous MCJ in the liver was almost undetectable (FIG. 7). MCJ levels remained very low for at least 7 days, and even after 9 days of administration no complete recovery of MCJ levels was seen (FIG. 7). Therefore, siMCJ was very efficient in suppressing MCJ protein expression for long periods of time.

Administration of siMCJ was conducted to study the effect of siMCJ in DILI treatment using an acetaminophen (APAP) mouse model that prevents acetaminophen-induced liver injury. The results described herein above indicate that MCJ expression can be targeted by administration of siMCJ. Thus, further studies were conducted to test whether a single siMCJ administration could protect the liver from damage caused by acetaminophen administration. Wild type mice were given a dose of siMCJ along with in vivofectamine. Control mice did not receive siMCJ. Next, 20 hours later, all mice received an intraperitoneal dose of acetaminophen as described above. Mice were collected 24 hours after acetaminophen administration. The level of MCJ in the liver was examined by Western blot analysis. MCJ levels in APAP mice treated with siMCJ were significantly reduced compared to APAP mice that did not receive siMCJ (FIG. 8A). It was already clear based on liver color that mice treated with siMCJ could be protected from acetaminophen-induced damage at the time of collection (the color in the liver was lighter after acetaminophen administration). These observations were confirmed by histological analysis of liver tissue sections, where livers from APAP mice show areas of obvious tissue damage, which are APAP mice treated with siMCJ. It was hardly detected in the liver of origin (FIG. 8B). Quantification of the damaged area in the liver provided an indication of the protective effect of siMCJ from acetaminophen-induced liver injury (FIG. 8C). Therefore, blocking MCJ expression with siMCJ in vivo protects against the damaging effects of acetaminophen in the liver.

Treatment with siMCJ after acetaminophen prevented acetaminophen-induced liver injury Currently an intensive care unit with acute liver failure due to overdose of acetaminophen (second major cause of acute liver failure) The only treatment for patients admitted to the hospital is N-acetylcysteine (NAC). NAC has a hepatoprotective effect, but only if administered within 8 hours after acetaminophen uptake. After 8 hours, the effect of NAC is known to be minimal and the only choice is liver transplantation. To determine if siMCJ can be used as a treatment in cases where the patient is after a longer period after acetaminophen overdose, mice are first treated with a high dose of Acetaminophen was administered. Subsequently, 24 hours later, intravenous injection with siMCJ and Invivofectamine was given to a cohort of mice (siMCJ mice). After 24 hours (48 hours after acetaminophen administration), mice were collected and the level of MCJ in the liver was determined by Western blot analysis, and the level of transaminase in the blood was measured as a diagnosis for liver failure. The level of MCJ in the liver in mice treated with siMCJ was almost undetectable (FIG. 9A), demonstrating efficacy in eliminating the presence of this protein in the liver. More importantly, the level of ALT (one of the transaminases measured in patients) was dramatically lower in mice treated with siMCJ (FIG. 9B).

Example 3
Human siMCJ was tested in human MCF7 cell line in vitro. Transfection of human cell line cells with a small amount of h-siMCJ (SEQ ID NO: 21) was sufficient to knock down MCJ protein expression (FIG. 10), whereas another mitochondrial protein, CoxIV. The level of was not affected. The transfection method involved excising human MCF7 cells transfected with h-siMCJ (5 nM), 30 hours later, cells were collected and MCJ expression was determined by Western blot analysis using anti-human MCJ antibody. Inspected. CoxIV (mitochondrial protein) and GAPDH were also tested as controls. The results demonstrated the use of siRNA to reduce MCJ protein expression.

Equivalents While several embodiments of the present invention have been described and illustrated herein, one of ordinary skill in the art will perform the functions and / or the results and / or benefits described herein. Various other means and / or structures for obtaining one or more are readily envisioned, and each such modification and / or modification is considered to be within the scope of the invention. More generally, those of ordinary skill in the art will appreciate that all parameters, dimensions, materials and shapes described herein are intended to be exemplary, and that actual parameters, dimensions, materials and / or shapes are It will be readily appreciated that the teaching (s) depends on the particular application (s) used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the embodiments described above are provided by way of example only, and, within the scope of the appended claims and their equivalents, the invention is described in a manner different from that specifically described and claimed. It will be appreciated that can be implemented in The present invention is directed to each individual feature, system, article, material, and / or method described herein. Further, any combination of two or more such features, systems, articles, materials and / or methods may be used if such features, systems, articles, materials and / or methods are consistent with each other. Included in range.

  All definitions are to be understood as governing the lexical definitions, definitions in documents incorporated by reference, and / or the ordinary meaning of the defined terms, as defined and used herein.

  The indefinite articles “a” and “an” as used herein and in the claims, as used herein, unless stated to the contrary, “at least one Should be understood to mean.

  The phrase “and / or” as used herein and in the claims, as used herein, is present in such coordinated elements, that is, in some cases as a bond, It should also be understood to mean “one or both” of the elements that are present separately. Other elements than those specifically identified by the “and / or” section, whether or not related to those elements specifically identified, unless clearly indicated to the contrary May optionally be present.

  All references, patents and patent applications and publications cited or referenced in this application are hereby incorporated by reference in their entirety.

Claims (58)

  A method of treating a drug-borne disease or condition in a subject comprising administering an MCJ-modulating compound to a subject in need of such treatment in an amount effective to treat the drug-induced disease or condition in the subject. .   2. The method of claim 1, wherein the MCJ modulating compound is an MCJ inhibitor compound that reduces MCJ polypeptide activity in the subject.   3. The method of claim 2, wherein reducing MCJ polypeptide activity comprises reducing one or more of MCJ polypeptide levels or activities.   4. The drug-borne disease or condition is one or more of drug-induced liver disease or condition and kidney disease or condition, heart disease or condition, and cardiovascular disease or condition. the method of.   The method according to any one of claims 1 to 4, wherein the disease or condition is an acute disease or condition.   The method according to any one of claims 1 to 4, wherein the disease or condition is a chronic disease or condition.   7. The method of any one of claims 1-6, wherein the MCJ modulating compound comprises one or more of an MCJ molecule, an anti-MCJ polypeptide antibody or functional fragment thereof, and a small molecule MCJ inhibitor.   8. The method of claim 7, wherein the MCJ modulating compound further comprises a targeting agent, optionally a mitochondrial targeting agent.   9. The method of claim 7 or 8, wherein the MCJ molecule is a mutant MCJ polypeptide or a polynucleotide encoding a mutant MCJ polypeptide.   Small molecule MCJ inhibitors are small interfering RNA molecules (siRNA), small hairpin RNA (shRNA) molecules, antisense DNA oligos, small guide RNA (sgRNA) molecules, transcription activator-like effector nuclease (talen) molecules The method according to claim 7 or 8.   12. The method of claim 10, wherein the siRNA molecule comprises the nucleic acid sequence set forth herein as SEQ ID NO: 21.   11. The method of claim 10, wherein the siRNA molecule comprises the nucleic acid sequence set forth herein as SEQ ID NO: 7.   The MCJ modulating compound is administered in a pharmaceutical composition, the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, optionally comprising one or more of a carrier agent, a delivery agent, a labeling agent, and a targeting agent. The method according to claim 1, comprising:   14. The method of claim 13, wherein the carrier agent comprises one or more of nanocarriers, cell penetrating peptides, polymers, dendrimers, siRNA bioconjugates, and lipid-based siRNA carriers.   14. The method of claim 13, wherein the pharmaceutical composition further comprises a drug known or suspected to induce a drug-borne disease or condition.   Drug-induced disease or condition is drug-induced cirrhosis, liver fibrosis, venous obstructive liver disease, idiosyncratic toxicity, Budd-Chiari syndrome, liver injury, kidney injury, drug allergy, acute kidney injury (AKI), fulminant 16. A method according to any one of the preceding claims comprising one or more of hepatitis, cholestasis, cardiotoxicity and alcohol intake.   17. The method of claim 16, wherein the drug-induced disease or condition inducing drug comprises one or more of ethanol, a pharmaceutical agent, or a biological agent.   18. The method of claim 17, wherein the inducing drug enters the subject by means of ingestion, the ingestion means optionally including one or more of inhalation, injection, absorption, infusion, infusion, drinking, and feeding.   Drugs are statins, antidepressants, antibiotics, benzodiazepines, nicotinic acid, tacrine, aspirin, quinidine, NSAID, aspirin, indomethacin, ibuprofen, naproxen, piroxicam, nabumetone, acetaminophen, phenytoin, isoniazid, diclofenac, augmentin, amoxicillin / Clavulanic acid combination, minocycline, nitrofurantoin, fenofibrate, methamphetamine, amphetamine, erythromycin, chlorpromazine, cotrimoxazole, sulfamethoxazole and trimethoprim combination, amitriptyline, temazepam, diazepam, carbamazepine, ampicillin, rifampin , Estradiol, captopril, oral contraceptive pill, oral contraceptive, protein Anabolic steroids, disulfiram, vitamin A, haloperidol, imipramine, tetracycline, phenytoin, methotrexate, amiodarone, methyldopa, chemotherapeutic agent, contrast agent, thiazine, phenothiazine, chloramphenicol, digoxin, digitoxin, oxazepam, phenobarbital, quinidine, vancomycin 18. The method of claim 17, comprising theophylline, verapamil, interferon, interferon beta 1a, and warfarin.   18. A method according to claim 17, wherein the biological agent comprises an herbal agent.   21. The method of claim 20, wherein the herbal agent comprises one or more of ephedula, birch, chaparral, valerian, cypress extract, birch extract, and birch leaf.   Drug disease or condition is overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia, steatosis, hepatic steatosis, cardiac steatosis, kidney Steatosis, muscular steatosis, abetalipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; liver disease, hepatitis, hepatitis, steatohepatitis, hepatitis C, genotype 3 hepatitis C, Alpha 1-antitrypsin deficiency, acute fatty liver of pregnancy, Wilson disease; kidney disease; heart disease, hypertension, ischemia, heart failure, cardiomyopathy; addiction; HIV; neurodegenerative disease, Parkinson's disease, Alzheimer's disease; or cancer metabolism 22. A method according to any one of claims 1 to 21 which is not a sexual disease or condition.   Subject is overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia, steatosis, hepatic steatosis, cardiac steatosis, renal steatosis, muscle Steatosis, abetalipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; liver disease, hepatitis, hepatitis, steatohepatitis, hepatitis C, genotype 3 hepatitis C, alpha 1-anti Trypsin deficiency, acute fatty liver of pregnancy, Wilson disease; kidney disease; heart disease, hypertension, ischemia, heart failure, cardiomyopathy; poisoning; HIV; neurodegenerative disease, Parkinson's disease, Alzheimer's disease; and cancer metabolic disease or condition 22. A method according to any one of claims 1 to 21, wherein the method does not have one or more of:   2. The method of claim 1, wherein the MCJ modulating compound is administered to the subject one or more before, during and after ingestion by the subject of a drug suitable for inducing a drug-induced disease or condition. .   A method of reducing a drug-borne disease or condition in a cell, comprising contacting the cell with an MCJ inhibitor compound in an amount effective to reduce MCJ polypeptide activity in the cell.   26. The method of claim 25, wherein reducing MCJ polypeptide activity comprises reducing one or more of the level or function of MCJ polypeptide in a cell.   27. The method of claim 25 or 26, wherein the cell is in vitro, ex vivo, or in vivo.   28. The method of claim 27, wherein in vivo cells are present in and contacted with the subject comprising administering to the subject an MCJ inhibitor compound.   29. The method of any one of claims 25-28, wherein the MCJ inhibitor compound comprises an MCJ molecule, an anti-MCJ polypeptide antibody or functional fragment thereof, or a small molecule MCJ inhibitor.   30. The method of claim 29, wherein the MCJ inhibitor compound further comprises a targeting agent, optionally a mitochondrial targeting agent.   31. The method of claim 29 or 30, wherein the MCJ molecule is a mutant MCJ polypeptide or a polynucleotide encoding a mutant MCJ polypeptide.   31. The method of claim 29 or 30, wherein the small molecule MCJ inhibitor is a siRNA molecule.   35. The method of claim 32, wherein the siRNA molecule comprises the nucleic acid sequence set forth herein as SEQ ID NO: 21.   34. The method of claim 32, wherein the siRNA molecule comprises the nucleic acid sequence set forth herein as SEQ ID NO: 7.   The cell is contacted with the MCJ inhibitor compound in the pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, and optionally one or more of the carrier agent, delivery agent, labeling agent, and targeting agent or 35. A method according to any one of claims 25 to 34, comprising a plurality.   36. The method according to any one of claims 25 to 35, wherein the drug-induced disease or condition is one or more of drug-induced liver disease or condition and kidney disease or condition, drug-induced heart disease or condition.   37. The method of any one of claims 25-36, wherein the drug-borne disease or condition is one or more of an acute disease or condition and a chronic disease or condition.   38. The method of claim 37, wherein the carrier agent comprises one or more of nanocarriers, cell permeable peptides, polymers, dendrimers, siRNA bioconjugates, and lipid-based siRNA carriers.   38. The method of claim 37, wherein the pharmaceutical composition further comprises a drug known or suspected to induce a drug-borne disease or condition.   Drug-induced disease or condition is drug-induced cirrhosis, liver fibrosis, venous obstructive liver disease, idiosyncratic toxicity, Budd-Chiari syndrome, liver injury, kidney injury, drug allergy, acute kidney injury (AKI), fulminant 40. The method of any one of claims 25-39, comprising one or more of hepatitis, cholestasis, cardiotoxicity, and alcohol intake.   40. The method of any one of claims 25-39, wherein the drug-induced disease or condition inducing drug comprises one or more of ethanol, a pharmaceutical agent, or a biological agent.   41. The method of claim 40, wherein a drug-induced disease or condition is induced in a cell by contacting the induced drug with the cell.   Drugs are statins, antidepressants, antibiotics, benzodiazepines, nicotinic acid, tacrine, aspirin, quinidine, NSAID, aspirin, indomethacin, ibuprofen, naproxen, piroxicam, nabumetone, acetaminophen, phenytoin, isoniazid, diclofenac, augmentin, amoxicillin / Clavulanic acid combination, minocycline, nitrofurantoin, fenofibrate, methamphetamine, amphetamine, erythromycin, chlorpromazine, cotrimoxazole, sulfamethoxazole and trimethoprim combination, amitriptyline, temazepam, diazepam, carbamazepine, ampicillin, rifampin , Estradiol, captopril, oral contraceptive pill, oral contraceptive, protein Anabolic steroids, disulfiram, vitamin A, haloperidol, imipramine, tetracycline, phenytoin, methotrexate, amiodarone, methyldopa, chemotherapeutic agent, contrast agent, thiazine, phenothiazine, chloramphenicol, digoxin, digitoxin, oxazepam, phenobarbital, quinidine, vancomycin 41. The method of claim 40, comprising: theophylline, verapamil, interferon, interferon beta 1a, and warfarin.   41. The method of claim 40, wherein the biological agent comprises an herbal extract.   41. The herbal extract comprises one or more of ephedula, birch, Comfrey, germander, chaparral, valerian, cypress extract, birch extract, and birch leaf. the method of.   Drug disease or condition is overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia, steatosis, hepatic steatosis, cardiac steatosis, kidney Steatosis, muscular steatosis, abetalipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; liver disease, hepatitis, hepatitis, steatohepatitis, hepatitis C, genotype 3 hepatitis C, Alpha 1-antitrypsin deficiency, acute fatty liver of pregnancy, Wilson disease; kidney disease; heart disease, hypertension, ischemia, heart failure, cardiomyopathy; addiction; HIV; neurodegenerative disease, Parkinson's disease, Alzheimer's disease; or cancer metabolism 46. The method of any one of claims 25 to 45, wherein the method is not a sexual disease or condition.   Cells are overweight, weight gain, obesity, non-alcoholic fatty liver disease, diabetes, insulin resistance, alcoholic fatty liver disease, dyslipidemia, steatosis, hepatic steatosis, cardiac steatosis, renal steatosis, muscle Steatosis, abetalipoproteinemia, glycogenosis, Weber Christian disease, lipodystrophy; liver disease, hepatitis, hepatitis, steatohepatitis, hepatitis C, genotype 3 hepatitis C, alpha 1-anti Trypsin deficiency, acute fatty liver of pregnancy, Wilson disease; kidney disease; heart disease, hypertension, ischemia, heart failure, cardiomyopathy; poisoning; HIV; neurodegenerative disease, Parkinson's disease, Alzheimer's disease; and cancer metabolic disease or condition 47. The method of any one of claims 25 to 46, wherein the method does not have one or more of:   26. The method of claim 25, wherein the cell is contacted with the MCJ inhibitor compound one or more before, during and after contacting the cell with a drug suitable for inducing a drug-borne disease or condition.   A composition comprising an MCJ inhibitor compound and a pharmaceutically acceptable carrier.   50. The composition of claim 49, further comprising one or more of a carrier agent, a delivery agent, a labeling agent, and a targeting agent.   51. The composition of claim 50, wherein the carrier agent comprises one or more of nanocarriers, cell penetrating peptides, polymers, dendrimers, siRNA bioconjugates, and lipid-based siRNA carriers.   51. The composition of claim 50, wherein the pharmaceutical composition further comprises a drug known or suspected to induce a drug-borne disease or condition.   53. The composition according to any one of claims 49 to 52, wherein the MCJ inhibitor compound comprises a targeting agent, optionally a mitochondrial targeting agent.   54. The composition according to any one of claims 49 to 53, wherein the MCJ inhibitor compound comprises a small molecule MCJ inhibitor.   Small molecule MCJ inhibitors are small interfering RNA molecules (siRNA), small hairpin RNA (shRNA) molecules, antisense DNA oligos, small guide RNA (sgRNA) molecules, transcription activator-like effector nuclease (talen) molecules 55. The composition of claim 54.   56. The composition of claim 55, wherein the small molecule MCJ inhibitor molecule has a nucleic acid sequence set forth herein as SEQ ID NO: 21.   56. The composition of claim 55, wherein the small molecule MCJ inhibitor molecule has a nucleic acid sequence described herein as SEQ ID NO: 7.   50. The composition of claim 49, further comprising a drug suitable for inducing a drug-induced disease or condition in at least one of the cell and the subject, respectively, when the drug contacts the cell or is delivered to the subject. .